<![CDATA[
<body bgcolor="ffffff">
<a name="top">
<table border=3>
<tr><td><IMG SRC="../images/h_ana.gif" ALT="Anatomy"></td>
</table><p>
<table>
<tr><td valign="top" align=left>© Neuroscience-Net<br> Volume 1, Article #10003
<td width=500 align=right>Received May 12, 1996<br>
Accepted for Publication June 25, 1996<br>
Published July 9, 1996<br>
</table><br>
<A HREF="index.html"><IMG SRC="b_back.gif" ALT="Back" Border=0 Align="LEFT"></A>
<CENTER>
<A HREF="../../articles.html"><IMG SRC="b_ita1.gif" ALT="Articles Index" Border=0></A>
<A HREF="../../index.html"><IMG SRC="b_home1.gif" ALT="Home Page" Border=0></A>
</CENTER><P>
<center>
<font size=+1><b>Brain-derived Neurotrophic Factor and S100<IMG SRC="../images/beta.gif"
ALT="beta">: Trophic Interactions on Cultured Serotonergic Neurons
</b></font><p>
</center>
Running Title: Trophic interactions between BDNF and S100<SUB><IMG SRC="../images/beta.gif"
ALT="beta"></SUB><P>
Mayumi Nishi<SUP>1</SUP>, Jose C. Poblete<SUP>1</SUP>, Patricia M. Whitaker-Azmitia<SUP>2</SUP>, and Efrain C. Azmitia<SUP>1</SUP> <SUP>1</SUP>. Dept. of Biology, New York University, NY, <SUP>2</SUP> Dept. of Psychiatry, SUNY, Stony Brook.<P>
Send correspondence to:<BR>
Dr. Efrain C. Azmitia<BR>
Dept. of Biology, New York University<BR>
1009 Main Building , 100 Washington Square<BR>
New York, NY 10003<BR>
(212) 998-8235<BR>
FAX: (212) 995-4175<BR>
E-mail: <A HREF="mailto:azmitia@acf2.nyu.edu">azmitia@acf2.nyu.edu</A><P>
<CENTER><STRONG>Key Words:</STRONG> BDNF, 5-HT, S100<SUB><IMG
SRC="../images/beta.gif" ALT="beta"></SUB>, Raphe, Primary culture, Glia, Differentiation , Morphometric
analysis, dynamic instability.</CENTER><P>
<ul>
<li><A HREF="#intro">Introduction</A>
<li><A HREF="#materials">Materials and Methods</A>
<li><A HREF="#results">Results</A>
<li><A HREF="#discuss">Discussion</A>
<li><A HREF="#legends">Legends</A>
<li><A HREF="#acknowledge">Acknowledgements</A>
<li><A HREF="#references">References</A>
</ul>
<HR>
<A NAME="intro"></A>
<FONT SIZE=+2><STRONG>Introduction</STRONG></FONT><P>
While many protein neurotrophic factors are present in the central nervous system (CNS) and play important
roles in neural development, differentiation and survival, few have been found to have trophic activity on
serotonergic neurons. The most potent protein factors on serotonergic growth thus far identified are
S100<SUB><IMG SRC="../images/beta.gif" ALT="beta"></SUB> (Azmitia et al., 1990; Liu and Lauder,
1992) and BDNF (Eaton et al., 1995; Mamounas et al.,1995; White et al.,1994). S100<SUB><IMG
SRC="../images/beta.gif" ALT="beta"></SUB>, a calcium-binding protein synthesized and stored in CNS glial
cells, acts mainly on neurite extension (Azmitia et al., 1990; Kligman and Marshak, 1985) by stabilization of
microtubules and inhibition of PKC phosphorylation of GAP-43, MAP-2 and tau (Hesketh and Baudier,
1986; Baudier et al, 1987; Sheu et al, 1994). It has been shown that the release of S100<SUB><IMG
SRC="../images/beta.gif" ALT="beta"></SUB> from glial cells is induced by 5-HT1A agonists (Whitaker-
Azmitia et al., 1990). <P>
In contrast, BDNF, derived mainly from neurons, promotes survival and synthesis of serotonin in an
immortalized serotonergic neuronal cell line without an increase in 5-HT reuptake (Eaton et al.,1995). Direct
cortical infusion of BDNF to para-chloroamphetamine (PCA) treated rats, produced an increase in sprouting,
5-HT synthesis and 3H-5-HT uptake (Mamounas et al., 1995). BDNF effects on serotonergic neurons may
result from the direct action of BDNF on serotonergic neurons via trkB receptors with a cytoplasmic tyrosine
kinase domain (gp145trkB) (Klein et al., 1991) expressed on raphe neurons (Merlio et al., 1992; White et al.,
1994). <P>
The direct action of BDNF on sprouting of serotonergic axons persists for months after the end of treatment
(Mamounas et al., 1995b). This long-term trophic action of BDNF may depend on the activation of the
trophic activity of glial cells. Recently, it has been shown that cultured cortical glial cells, which predominantly
express gp95trkB lacking tyrosine kinase domain (Frisen et al.,1993), also respond to BDNF (Roback et
al.,1995). To test the hypothesis that some of the trophic effects exerted by BDNF on serotonergic neurons are
mediated via the glial protein S100<SUB><IMG SRC="../images/beta.gif" ALT="beta"></SUB>, we
investigated the effects of BDNF on S100<SUB><IMG SRC="../images/beta.gif" ALT="beta"></SUB>
immunoreactivity, effects of BDNF and S100<SUB><IMG SRC="../images/beta.gif" ALT="beta"></SUB>
on serotonergic properties, and the actions of anti- S100<SUB><IMG SRC="../images/beta.gif"
ALT="beta"></SUB> antibody on these BDNF effects in primary raphe cultures.<P>
<HR>
<A NAME="materials"></A>
<FONT SIZE=+2><STRONG>Materials and Methods</STRONG></FONT><P>
Primary Culture<BR>
Dissociated raphe primary cultures were prepared from 14-day-old Sprague-Dawley rat fetuses according to
the method of Azmitia and Hou (1994). Briefly, pathogen-free timed pregnant Sprague-Dawley rats were
obtained from Taconic Farms (Germantown, NY) to arrive for use on gestation (day 14). The rats were
anesthetized with CO<sub>2</sub>, swabbed with 70% ethanol, and the fetuses were removed by cesarean
procedure using antiseptic procedure. The fetuses were removed from placenta in a laminar flow hood and
transferred to ice-cold D1 solution (0.8% NaCl, 0.04% KCl , 0.006%
Na<sub>2</sub>HPO4.1<sub>2</sub>H<sub>2</sub>O, 0.003% KH<sub>2</sub>PO4, 0.5% glucose,
0.00012% phenol red, 0.0125% penicillinG, and 0.02%streptomycin). Whole brains were removed from the
skull, and then a coronal cut was made at the apex of the mesencephalic flexure and at the pontine flexure. The
tegmentum was exposed by cutting dorsally from the cerebral aqueduct and separating the corpus
quadrigeminal. Two sagittal cuts were made approximately 0.5-1.0 mm lateral to the midline. The isolated
brain pieces were mechanically dissociated by triturating through a fire-polished glass pipette. The use of trypsin
was avoided since a recovery of large neurons following trypsin dissociation was reduced. The cells were
plated at an initial plating density of 8x10<sup>5</sup> cells/cm<sup>2</sup> by adding 200 ul of the cell
suspension to each well (area of 0.28 cm<sup>2</sup>; Lab-Tek Chamber Slide, Nunc, Inc., IL) precoated
with 15 mg/ml poly-D-lysine (MW=70,000-150,000; Sigma, MO). The cultures were maintained in complete
neuronal medium (CNM), consisting of 92.5% (v/v) Eagle's minimum essential medium (MEM, Sigma), 1%
(w/v) non-essential amino acids (Gibco-Life Technologies, Inc., MD), 0.16% (w/v) glucose and 5 % (v/v) fetal
bovine serum (Sigma) in a CO<sub>2</sub> incubator at 37 <sup>o</sup>C with 5%
CO<sub>2</sub>/95% air. <P>
Growth factor treatment<BR>
After 24h the CNM was removed and the cultures were rinsed with serum-free medium without serotonin
and steroids (MEM with 0.16% of glucose, 1% non-essential amino acids, 20 mM putrescine, 15 nM sodium
selenite, 5 mg/ml insulin, and 100 mg/ml transferrin, 5 mgml bovine serum albumine (BSA)). At this time point,
cultures were supplemented with human recombinant BDNF (50 ng/ml, Promega), S100<SUB><IMG
SRC="../images/beta.gif" ALT="beta"></SUB>(10 ng/ml, 99% homogeneous by SDS-PAGE, East Acres
Biologicals, MA), BDNF (50 ng/ml) + S100<SUB><IMG SRC="../images/beta.gif" ALT="beta"></SUB>
(10 ng/ml) or BDNF (50 ng/ml) + rabbit anti-S100<SUB><IMG SRC="../images/beta.gif"
ALT="beta"></SUB> antibody (1/10,000 dilution of purified IgG fraction, a gift from Dr. L. VanEldik).
BDNF and S100<SUB><IMG SRC="../images/beta.gif" ALT="beta"></SUB> were prepared in a sterile
solution of phosphate buffered saline (PBS) with 5 mg/ml BSA. As a control antibody, normal rabbit IgG (1/
10,000 dilution) was used, because we could not get preimmune serum. These concentrations were chosen
based on effective ranges cited in the literature. Generally, we prepared three or four culture wells for each
experimental condition in each experiment. Before fixation of the cells for immnucytochemistry, total numbers of
living cells per well was measured by using trypan blue staining from one culture well for each experimental
group.<P>
Immunocytochemistry<BR>
After four days of incubation, cultured cells were fixed for 15 min at 37oC in 4 % of paraformaldehyde in
phosphate buffer (PB, pH 7.4). After rinsing with PB, the cells were blocked with 2 % of bovine serum
albumine in PB for 1 h at room temperature (RT). The fixed cells were incubated with rabbit antisera directed
against 5-HT (1/4,000 dilution, Incstar; MI) or S100<SUB><IMG SRC="../images/beta.gif"
ALT="beta"></SUB> (1/10,000 dilution) in PB containing 2% BSA for 48 h at 4oC. After rinsing with PB
containing 2% BSA five times, the cultured cells were reacted with biotinylated goat anti-rabbit antibody
(1/250 dilution; Boehringer Mannheim, IN) for 1 h at RT. The cultures were rinsed with PB five times and
reacted with avidin-biotin-peroxidase complex (Vector Laboratories, CA) for 1h at RT. The cells were
visualized with 0.02% 3,3'-diaminobenzidine (Sigma) and 0.006% H<sub>2</sub>O<sub>2</sub> in Tris-
HCl buffered saline (pH 7.6). Slides were dehydrated with ethanol, followed by xylene, and cover-slipped in
Permount. As a negative control, the entire immunocytochemical procedure was replicated, eliminating primary
antibody. Nonspecific staining was not observed. <P>
Morphometric Analysis<BR>
Slides were subjected to morphometric analyses according to the previously described method (Azmitia et
al., 1995; Nishi et al., 1996). Briefly, the Optimas (ver.4.0) image acquiring software program (Bioscan, Inc.,
WA) was used for all morphometric analyses. Using a Leitz Microscope equipped with a video camera, the
cells were captured randomly. S100<SUB><IMG SRC="../images/beta.gif" ALT="beta"></SUB> -IR
intensity was determined by measuring the mean area gray-value (ArGV) of each cell (magnification for this
procedure: 40 x objective). The background light was standardized to a ArGV of 100. Since a higher ArGV
corresponds to a lighter area, the measurements were subtracted from 100 in order to facilitate graphic analysis.
The value of 100-ArGV obtained from 600 cells from three independent experiments (200 cells from each
experiment) was calculated and presented as mean value + SEM and a histogram. For the analysis of soma
area of 5-HT-IR neurons, the perimeter of each cell body was outlined with cursor using the automatic edging
feature which detects differences in light intensity and subjected to Optimas area measurement. For the analysis
of total process length for individual 5-HT-IR neurons, all primary processes are captured and subjected to
Optimas line-length measurement. In the analyses of soma area and length, magnification was 63x objective.
For each condition, about 600 cells were measured from three independent experiments (200 cells from each
experiment). <P>
Statistical analysis:<BR>
The statistical significance of all quantitative data was determined by analysis of variance (ANOVA).
Comparison of differences between individual means were tested using the Tukey honest difference
method.<P>
<HR>
<A NAME="results"></A>
<FONT SIZE=+2><STRONG>Results</STRONG></FONT><P>
Descriptive analysis of BDNF and S100<SUB><IMG SRC="../images/beta.gif" ALT="beta"></SUB> on rat
brainstem raphe cultures<P>
Raphe neurons were prepared from E14 rats and grown in serum free media without serotonin or steroids.
At the given cell density and our serum-free media, cultured cells were about 75 % of microtubule-associated
protein 2 (MAP2) positive neurons and about 25 % of S100<SUB><IMG SRC="../images/beta.gif"
ALT="beta"></SUB> positive glial cells after 4 days of culture (data not shown). The effects of BDNF
(50ng/ml) or S100<SUB><IMG SRC="../images/beta.gif" ALT="beta"></SUB> (10 ng/ml) on the survival
and morphological differentiation of 5-HT-IR neurons were examined in 4-day-old cultures. Also the effect of
BDNF on S100<SUB><IMG SRC="../images/beta.gif" ALT="beta"></SUB> immunoreactivity was
analyzed. <A HREF="#fig.1">Figures 1</A> (low magnification) and <A HREF="#fig.2">2</A> (high
magnification) showed a typical morphology of 5-HT-IR neurons in each treatment group. As shown in the
control culture (Figs.1A and 2A), 5-HT-IR cells had fusiform shape, several stained processes and comprised
about 0.6% of total cells.
In the culture treated with BDNF (<A HREF="#fig.1">Figs 1C</A> and <A HREF="#fig.2">2C</A>)
and BDNF+S100<SUB><IMG SRC="../images/beta.gif" ALT="beta"></SUB> (<A HREF="#fig.1">Figs
1D</A> and <A HREF="#fig.2"> 2D</A>), the 5-HT-IR cells had larger soma areas, shorter processes and
increased 5-HT-immunoreactivity as compared to control cultures. The soma shape in these BDNF-neurons
was more round and oval than fusiform. In the culture treated with S100<SUB><IMG
SRC="../images/beta.gif" ALT="beta"></SUB> (<A HREF="#fig.1">Figs 1B</A> and <A HREF="#fig.2">
2B</A>), 5-HT-IR neurons showed more and longer processes compared to both control and BDNF-treated
cultures. There was no increase in 5-HT-IR staining or soma area compared to control culture. The neurons
had a multipolar shape with numerous processes emanating from the soma.<P>
BDNF promotes S100<SUB><IMG SRC="../images/beta.gif" ALT="beta"></SUB> immunoreactivity in
glial cells <P>
In order to investigate whether exogenous BDNF directly affects S100<SUB><IMG
SRC="../images/beta.gif" ALT="beta"></SUB> level in glial cells, S100<SUB><IMG
SRC="../images/beta.gif" ALT="beta"></SUB> immunoreactivity was examined in raphe cultures treated with
BDNF for 3 h or 2 days. In BDNF-treated cultures, S100<SUB><IMG SRC="../images/beta.gif"
ALT="beta"></SUB> immnuoreactivity was stimulated after 3h (<A HREF="#fig.3"> Fig. 3</A>) and 2 days
(data not shown) of treatment. Particularly, in 3h BDNF-treated cultures, S100<SUB><IMG
SRC="../images/beta.gif" ALT="beta"></SUB> immunoreactivity was increased within and around the
astrocytes. In BDNF-treated glial cells, there was an increased appearance of membrane blebbing showing
S100<SUB><IMG SRC="../images/beta.gif" ALT="beta"></SUB>-IR. Quantitative image analysis indicated
that BDNF produced a 53 % increase (p<0.05) in S100<SUB><IMG SRC="../images/beta.gif"
ALT="beta"></SUB> immunoreactivity as compared to control values and also shifted the distribution of
S100<SUB><IMG SRC="../images/beta.gif" ALT="beta"></SUB>-IR upward (<A HREF="#fig.4">
Fig.4</A>). The increase in S100<SUB><IMG SRC="../images/beta.gif" ALT="beta"></SUB> seen in
raphe cultures was also observed in hippocampal cultures which contained no exogenous or intrinsic source of
5-HT (data not shown).<P>
Survival of 5-HT-IR neurons increased by BDNF but not S100<SUB><IMG SRC="../images/beta.gif"
ALT="beta"></SUB> in raphe cultures <P>
As shown in <A HREF="#fig.5">Figure 5</A>, BDNF treatment and combining BDNF treatment with
S100<SUB><IMG SRC="../images/beta.gif" ALT="beta"></SUB> for 3 days significantly increased the
number of 5-HT-IR cells (+78% and +68% of control value, respectively, p<0.05), whereas
S100<SUB><IMG SRC="../images/beta.gif" ALT="beta"></SUB> did not show a significant increase. Total
numbers of viable cells determined by trypan-blue in each well treated with BDNF, S100<SUB><IMG
SRC="../images/beta.gif" ALT="beta"></SUB>, a combination of BDNF and anti-S100<SUB><IMG
SRC="../images/beta.gif" ALT="beta"></SUB> antibody or rabbit IgG did not show significant differences as
compared to those observed in control cultures after 4 days of in vitro (data not shown).<P>
Morphological differentiation of 5-HT-IR neurons induced by BDNF or S100<SUB><IMG
SRC="../images/beta.gif" ALT="beta"></SUB><P>
The effects of BDNF or S100<SUB><IMG SRC="../images/beta.gif" ALT="beta"></SUB> on the soma
area and the total process length of 5-HT-IR neurons were examined in 4-day-old cultures. In BDNF-treated
cultures, the soma area of 5-HT-IR cells were significantly increased (+35%, p<0.05), while
S100<SUB><IMG SRC="../images/beta.gif" ALT="beta"></SUB> did not show a significant increase in the
soma area (<A HREF="#fig.6">Fig.6</A>). For analyses of the total length of 5-HT-IR processes, all the
processes branching directly from the cell body (primary process) were measured. BDNF showed no
significant change in the total length of primary processes for individual 5-HT-IR neurons (<A
HREF="#fig.7">Fig. 7</A>). In contrast, S100<SUB><IMG SRC="../images/beta.gif"
ALT="beta"></SUB> significantly promoted the total process length (+110%, P<0.01), especially, the length
of the longest process. As observed in , <A HREF="#fig.2">Fig.2B</A>S100<SUB><IMG
SRC="../images/beta.gif" ALT="beta"></SUB> treatment increased the number of 5-HT-IR processes.
Combined treatment of BDNF and S100<SUB><IMG SRC="../images/beta.gif" ALT="beta"></SUB>
induced an increase in the soma area (+34%, p<0.05) but not in the total process length (+20%, not significant).
<P>
Anti-S100<SUB><IMG SRC="../images/beta.gif" ALT="beta"></SUB> antibody blocks BDNF trophic-
actions on 5-HT-IR neurons<P>
The question of possible trophic interactions between BDNF and S100<SUB><IMG
SRC="../images/beta.gif" ALT="beta"></SUB> was addressed by using anti-S100<SUB><IMG
SRC="../images/beta.gif" ALT="beta"></SUB> antibody blocking method. Combining BDNF treatment with
anti-S100<SUB><IMG SRC="../images/beta.gif" ALT="beta"></SUB> antibody showed a decrease in the
number of 5-HT-IR cells as compared to BDNF treated cultures (-30%, p<0.05) (<A
HREF="#fig.1">Figs.1E</A>,<A HREF="#fig.2">2E</A> and <A HREF="#fig.5">5</A>). Also an
antibody to S100<SUB><IMG SRC="../images/beta.gif" ALT="beta"></SUB> blocked the effect of BDNF
on the soma area (-24% as compared to BDNF treatment, p<0.05) (Figs. <A HREF="#fig.1">1E</A>,<A
HREF="#fig.2">2E</A> and <A HREF="#fig.6">6</A>). In contrast, anti-S100<SUB><IMG
SRC="../images/beta.gif" ALT="beta"></SUB> antibody exerted no significant blocking effect on BDNF in
the total length of 5-HT-IR processes. The addition of control rabbit IgG did not block the effect of BDNF
on the increase in the survival nor the soma area of 5-HT-IR neurons. <P>
<HR>
<A NAME="discuss"></A>
<FONT SIZE=+2><STRONG>Discussion</STRONG></FONT><P>
It has been demonstrated that survival and maturation of serotonergic neurons are stimulated by BDNF
(Eaton et al.,1995; Mamounas et al., 1995). An immortalized serotonergic cell line, RN46A, was established
from embryonic day-13 rat raphe cells with a retrovirus encoding the temperature-sensitive mutant of SV40
(Whittemore and White, 1993). BDNF increases the number of 5-HT-IR and tryptophan hydroxylase-IR cells
in RN46A cultures under depolarizing condition (White et al.,1994) and stimulates RN46A cell survival and 5-
HT levels in differentiating conditions (Eaton et al., 1995). No effect on 3H-5-HT uptake was found. BDNF
administered in the cerebral cortex stimulates the sprouting of 5-HT fibers both in intact and 5-HT toxin-
lesioned rat (Mamounas et al.,1995). These authors noted an increase in the 5-HT levels and 3H-5-HT
uptake. The mechanism of action may involve activation of the trkB receptors located on 5-HT neurons (Merlio
et al.,1992; White et al., 1994).
The effects of BDNF on 5-HT sprouting persists for months after the end of treatment (Mamounas et al.,
unpublished data). One of the explanations for this log-term actions is that BDNF may affect some type of
supporting system. Here we have investigated the possible trophic interactions of BDNF with the glial protein
S100<SUB><IMG SRC="../images/beta.gif" ALT="beta"></SUB> in primary raphe cultures.<P>
The present study using primary dissociated cultures from rat raphe has shown that BDNF promoted the
survival of 5-HT-IR cells. In RN46A cells, long-term (8 days) exposure to BDNF requires depolarizing
condition for promoting the survival of 5-HT-IR cells, whereas short-term (4days) exposure to BDNF has a
significant effect on survival without depolarization (Eaton et al., 1995). These results are consistent with the
present results of primary raphe culture study with short-term (3 days) BDNF treatment. The survival promoting
effect caused by BDNF seems to be relatively specific to 5-HT-IR neurons, since BDNF did not produce a
significant increase in the total cell numbers in raphe cultures.<P>
It has been demonstrated that RN46A cells express significant levels of low-affinity neurotrophin receptor,
p75NTR, and the high-affinity BDNF receptor protein, trkB. The treatment with BDNF during differentiation
markedly increases the expression of both neurotrophin receptors (White et al.,1994), indicating that the
stimulation of RN46A cell survival with BDNF could be regulated by initial tyrosine phosphorylation of trkB.
The transduction mechanism in response to BDNF-induced trk receptor phosphorylation involve mitogen
activated protein (MAP; also termed microtubule associated protein) kinase (Marsh et al., 1993) and
phospholipase C <IMG SRC="../images/gamma.gif" ALT="gamma">1 (Widmer et al., 1992,1993), which
result in c-fos induction and Ca<sup>2+</sup> -mobilization. These signal transduction events may contribute to the
serotonergic neuronal survival. In contrast to BDNF, exogenously added S100<SUB><IMG
SRC="../images/beta.gif" ALT="beta"></SUB> did not affect the survival of 5-HT-IR cells in primary raphe
culture. <P>
Immunocytochemical studies showed that BDNF treatment for both 3 h and 2 days increased the
S100<SUB><IMG SRC="../images/beta.gif" ALT="beta"></SUB> level in glial cells. Especially, in the 3h
treatment cultures, S100<SUB><IMG SRC="../images/beta.gif" ALT="beta"></SUB>-IR was more intense
inside and around the glial cells. The pattern has been observed in vivo and may indicate released
S100<SUB><IMG SRC="../images/beta.gif" ALT="beta"></SUB> (Zhou et al, 1995). The astrocytes after
BDNF treatment show S100<SUB><IMG SRC="../images/beta.gif" ALT="beta"></SUB> in blebbs
evaginating from the plasma membrane. These results suggest that BDNF promotes the process of producing
and/or releasing of S100<SUB><IMG SRC="../images/beta.gif" ALT="beta"></SUB>. The mechanism of
BDNF effects on glial cells remains to be controversial. Recently Roback et al. (1995) has shown that cultured
cortical glial cells respond to BDNF. They showed that BDNF produced a marked increase in MAP kinase
activity, intracellular calcium concentration and c-fos expression in glial cells. It has been suggested that the
mechanism of BDNF-induced signal transduction in glial cells may involve relatively small amount of gp145trkB,
but involvement of the more abundant gp95trkB cannot be excluded. Since the present study employed the
neuronal and glial mixed raphe cultures, whether the increase in S100<SUB><IMG SRC="../images/beta.gif"
ALT="beta"></SUB> immunoreactivity is induced via the direct action of BDNF on glial cells or through the
release of 5-HT remains to be questioned. It has been shown that the release of S100<SUB><IMG
SRC="../images/beta.gif" ALT="beta"></SUB> from astrocytes is induced by 5-HT1A agonists (Whitaker-
Azmitia et al., 1990). In the adult, removal of 5-HT induces a reduction in S100<SUB><IMG
SRC="../images/beta.gif" ALT="beta"></SUB> (Azmitia et al., 1995; Haring et al., 1994), but an enhanced
stimulation of this protein by 5-HT1A agonists (Azmitia et al.,1995) . A previous study has shown that the
trophic actions of S100<SUB><IMG SRC="../images/beta.gif" ALT="beta"></SUB> do not require neuronal
support (Zhou et al., 1995). The current studies are examining if the BDNF-induced increase in
S100<SUB><IMG SRC="../images/beta.gif" ALT="beta"></SUB> immunoreactivity has a correlation with
the increase in 5-HT release from 5-HT-IR neurons and /or the direct action of BDNF on glial cells. Our
preliminary data showed that BDNF increased immunoreactivities of 5-HT and tryptophan hydroxylase, a rate
limiting enzyme converting tryptophan to 5-HT. In order to eliminate the effects of 5-HT, we employed the
primary rat hippocampal culture. BDNF also stimulated the S100<SUB><IMG SRC="../images/beta.gif"
ALT="beta"></SUB> immunoreactivity in hippocampal glial cells (unpublished data).<P>
It has been indicated that BDNF stimulates MAP kinase which can phosphorylate microtubule associated
proteins such as MAP2 and tau (Barbacid, 1994; Marsh et al., 1993). Furthermore, BDNF has been reported
to activate phospholipase C <IMG SRC="../images/gamma.gif" ALT="gamma">1 in cultured cortical neurons
and elevate protein kinase C (PKC) activity, which can also promote the phosphorylation of MAP2 and tau
(Widmer et al., 1993). Phosphorylation of microtubule associated proteins inhibits tubulin polymerization
causing breakdown of the cytoskeleton, which is important for the initialization of dendritic arborization (Diez-
Guerra and Avila, 1993; Nishida et al.,1987) and spine formation ( Aoki and Skovitz, 1988). In contrast,
S100<SUB><IMG SRC="../images/beta.gif" ALT="beta"></SUB> inhibits the phosphorylation of MAP2 by
PKC and that of tau by CaMKII, and promotes microtubule assembly and stability (Hesketh and Baudier,
1986; Baudier and Cole, 1988). S100<SUB><IMG SRC="../images/beta.gif" ALT="beta"></SUB> may
also induce neurite extension by regulating the phosphorylation by PKC of GAP43 (Sheu et al., 1994), an
important component of active growth cone, being involved in neurite outgrowth. S100<SUB><IMG
SRC="../images/beta.gif" ALT="beta"></SUB> is a potent neurite extension factor for cortical and
serotonergic neurons (Azmitia et al., 1990; Kligman and Marshak, 1985). There is a direct evidence that
S100<SUB><IMG SRC="../images/beta.gif" ALT="beta"></SUB> plays an active role in serotonergic
sprouting. The C6 glioma cell line, that secretes S100<SUB><IMG SRC="../images/beta.gif"
ALT="beta"></SUB>, transplanted into an adult rat hippocampus induces the sprouting of serotonergic fibers
(Ueda et al., 1995). <P>
The question of possible trophic interactions between BDNF and S100<SUB><IMG
SRC="../images/beta.gif" ALT="beta"></SUB> was addressed by using an antibody blocking approach. An
antibody to S100<SUB><IMG SRC="../images/beta.gif" ALT="beta"></SUB>, not normal rabbit IgG,
blocked the BDNF-induced increase in the survival of 5-HT-IR cells. This result suggests that trophic effects
of BDNF on the survival of 5-HT-IR neurons may require supporting actions of S100<SUB><IMG
SRC="../images/beta.gif" ALT="beta"></SUB> which is stimulated in raphe cultures treated with BDNF,
although an exogenously added S100<SUB><IMG SRC="../images/beta.gif" ALT="beta"></SUB> has no
effect on the survival. An antibody to S100<SUB><IMG SRC="../images/beta.gif" ALT="beta"></SUB>
also blocked the effects of BDNF on the soma area increase. It is indicated that BDNF increases the soma
area of 5-HT-IR neurons not only by stimulating S100<SUB><IMG SRC="../images/beta.gif"
ALT="beta"></SUB> which stabilizes cytoskeletal proteins such as MAP2 and tau, but also by promoting
kinase activities which might be essential for the initialization of arbolization and/or sprouting of neurites.
Together with the other results of the present study, this antibody blocking approach supported trophic
interactions between BDNF and S100<SUB><IMG SRC="../images/beta.gif" ALT="beta"></SUB>.<P>
BDNF stimulated the morphological differentiation by increasing the soma area of 5-HT-IR neurons
without increasing the neurite extension. S100<SUB><IMG SRC="../images/beta.gif" ALT="beta"></SUB>
stimulated the total length of 5-HT-IR processes without affecting the soma area. In particular, the length of the
longest neurite of each neuron was remarkably increased by S100<SUB><IMG SRC="../images/beta.gif"
ALT="beta"></SUB>. These results are consistent with the effects of Eaton et al (1995) with RN46A cells.
One possibility suggested by our experiments is that BDNF, synthesized by neurons, promotes the survival of
5-HT-IR neurons and stimulates the initialization of sprouting of 5-HT-IR neurons by enhancing the kinase
activities for cytoskeletal proteins, whereas S100<SUB><IMG SRC="../images/beta.gif"
ALT="beta"></SUB>, synthesized in glial cells, stimulates neurite extension by inhibiting kinase activities
(Fig.8). There is a lingering questions of why BDNF applied in cultures can stimulate S100<SUB><IMG
SRC="../images/beta.gif" ALT="beta"></SUB> but not result in process elongation, while S100<SUB><IMG
SRC="../images/beta.gif" ALT="beta"></SUB> applied alone does produce process elongation. In fact, when
we applied both BDNF and S100<SUB><IMG SRC="../images/beta.gif" ALT="beta"></SUB>, there was
no process outgrowth. As shown in the diagram(Fig.8), S100<SUB><IMG SRC="../images/beta.gif"
ALT="beta"></SUB> action (green line) would promote and stabilize neurite elongation, while BDNF
increases kinase activity (blue line) that would oppose this elongation process. In summary, the results indicate
that maximal trophic activity may require sequential, not concurrent, actions of BDNF and S100<SUB><IMG
SRC="../images/beta.gif" ALT="beta"></SUB>. Further studies are needed to explore this interaction.<P>
Our findings provide an important concept for neuronal-glial interactions that these two neurotrophic
factors, having different actions on signal transduction cascade, interact and support each other to exert
convergent effects on neuronal maturation. The current study is investigating more quantitative evidences using
5-HT uptake approach and also examining the differentiating effects of BDNF on some other serotonergic
properties. <P>
<A NAME="legends"></A><P>
<FONT SIZE=+2><STRONG>Legends:</STRONG></FONT><P>
<table border=5>
<tr><td><b>Please note, you can click on an image to view it in a much larger size.<br>
To navigate back to this page, please use the back feature that is on your browser.</b><br>
</table><p>
<A NAME="fig.1"></A><P>
<A HREF="ano3fo1l.html"> <IMG SRC="../images/an03f01s.jpg" ALT="Fig.1" HSPACE=10 VSPACE=10
BORDER=0><br clear=both>
<STRONG>Fig.1:</A> Effects of BDNF, S100<SUB><IMG SRC="../images/beta.gif"
ALT="beta"></SUB>, or BDNF + antiS100<SUB><IMG SRC="../images/beta.gif" ALT="beta"></SUB>
on the survival, the soma area, and the process length of 5-HT-IR neurons, low magnification.</STRONG>
Each photomicrograph shows a typical 5-HT-IR neurons in each treatment. Raphe cultures from E14 rats were
grown for 24h in the presence of fetal bovine serum, then medium was changed to serum free medium without
serotonin nor steroids, and treated with the following substances. A: Control culture was grown in the absence
of serum, serotonin nor steroids for the last 3 days before fixation. B: S100<SUB><IMG
SRC="../images/beta.gif" ALT="beta"></SUB> (10 ng/ml) was added for the last 3 days before fixation. C:
BDNF (50 ng/ml) was added for the last 3 days before fixation. D: BDNF (50 ng/ml) + S100<SUB><IMG
SRC="../images/beta.gif" ALT="beta"></SUB> (10 ng/ml) were added for the last three days before fixation.
E: BDNF (50ng/ml) + anti-S100<SUB><IMG SRC="../images/beta.gif" ALT="beta"></SUB> antibody
(1/10,000 dilution) were added for the last 3 days before fixation. After fixation, the cells were subjected to 5-
HT immunocytochemistry. In the cultures treated with BDNF (C) and BDNF + S100<SUB><IMG
SRC="../images/beta.gif" ALT="beta"></SUB> (D), the number of 5-HT-IR neurons were increased as
compared to control(A). Combining BDNF treatment with anti-S100<SUB><IMG SRC="../images/beta.gif"
ALT="beta"></SUB> antibody blocked the BDNF-induced increase in the survival of 5-HT-IR neurons (E).
Bar: 75um<P>
<A NAME="fig.2"></A><P>
<A HREF="ano3fo2l.html"> <IMG SRC="../images/an03f02s.jpg" ALT="Fig.2" WIDTH=144 HEIGHT=73
BORDER=0><br clear=both>
<STRONG>Fig.2</A> : Higher magnification of photomicrographs corresponding to those shown in
Fig.1.</STRONG> A: Control, B: S100<SUB><IMG SRC="../images/beta.gif" ALT="beta"></SUB> (10
ng/ml), C: BDNF (50ng/ml), D: BDNF (50ng/ml) + S100<SUB><IMG SRC="../images/beta.gif"
ALT="beta"></SUB> (10 ng/ml), E: BDNF (50 ng/ml) + anti-S100<SUB><IMG SRC="../images/beta.gif"
ALT="beta"></SUB> antibody (1/10,000 dilution). In the culture treated with BDNF (C) and BDNF +
S100<SUB><IMG SRC="../images/beta.gif" ALT="beta"></SUB> (D), more densely stained 5-HT-IR
neurons having larger soma area (asterisk) were observed. The treatment with S100<SUB><IMG
SRC="../images/beta.gif" ALT="beta"></SUB> promoted the extension of 5-HT-IR processes (arrow), but
not soma area (B). An antibody to S100<SUB><IMG SRC="../images/beta.gif" ALT="beta"></SUB>
blocked the increase in the soma area induced by BDNF (E). Bar: 35 micrometer
<P>
<A NAME="fig.3"></A><P>
<A HREF="ano3fo3l.html"> <IMG SRC="../images/an03f03s.jpg" ALT="Fig.3" WIDTH=144 HEIGHT=55
BORDER=0><br clear=both>
<STRONG>Fig.3</A> : Effects of BDNF on S100<SUB><IMG SRC="../images/beta.gif"
ALT="beta"></SUB> immunoreactivity in raphe culture.</STRONG> A: Control culture was grown in the
serum containing media for the first 24h, then in the absence of serum, serotonin nor steroids for the last 3 days
before fixation. B: BDNF (50 ng/ml) was added for the last 3 h before fixation. After fixation, the cells were
subjected to S100<SUB><IMG SRC="../images/beta.gif" ALT="beta"></SUB> immunocytochemistry. The
photomicrographs in Fig.4 represented the pseudocolor. In the culture treated with BDNF for 3 h,
S100<SUB><IMG SRC="../images/beta.gif" ALT="beta"></SUB> immunoreactivity was increased within
and around the astrocytes (arrow). In BDNF-treated glial cells, there was an increased appearance of
membrane blebbing showing S100<SUB><IMG SRC="../images/beta.gif" ALT="beta"></SUB>-IR. Bar: 55
micrometer
<P>
<A NAME="fig.4"></A><P>
<A HREF="ano3fo4l.html"> <IMG SRC="../images/an03f04s.gif" ALT="Fig.4" WIDTH=127 HEIGHT=144
BORDER=0><br clear="both">
<STRONG>Fig.4</A> : Quantitative analyses of BDNF effects on S100<SUB><IMG
SRC="../images/beta.gif" ALT="beta"></SUB> immunoreactivity.</STRONG> The levels of the respective
immunoreactivities of individual cells were quantified by morphometric analysis and sorted into five ranges of
increasing intensity (100-ArGV) from 0-20, 21-40, 41-60, 61-80, and 81-100. The values are mean (%) of
600 individual cells for each condition counted from three independent experiments.
<P>
<A NAME="fig.5"></A><P>
<A HREF="ano3fo5l.html"> <IMG SRC="../images/an03f05s.gif" WIDTH=144 HEIGHT=138
BORDER=0><br clear=both>
<STRONG>Fig.5</A> : Effects of BDNF (50 ng/ml), S100<SUB><IMG SRC="../images/beta.gif"
ALT="beta"></SUB> (10 ng/ml), BDNF(50 ng/ml) + S100<SUB><IMG SRC="../images/beta.gif"
ALT="beta"></SUB> (10 ng/ml), BDNF(50 ng/ml) + anti S100<SUB><IMG SRC="../images/beta.gif"
ALT="beta"></SUB> (1/10,000, dilution) and BDNF (50 ng/ml) + IgG (1/10,000, dilution) on the number of
5-HT-IR cells.</STRONG> The cells were treated for 3 days, at which time they were fixed and subjected to
5-HT immunocytochemistry. The mean values /well obtained from 3 independent experiments were calculated.
The results were analyzed using ANOVA followed by tukey honest test. Data were presented as the mean +
SEM. *: statistically significant (p<0.05) in comparison to the control value; **: statistically significant (p<0.05)
compared to the BDNF treated group.
<P>
<A NAME="fig.6"></A><P>
<A HREF="ano3fo6l.html"> <IMG SRC="../images/an03f06s.gif" ALT="Fig.6" Border=0><br clear=both>
<STRONG>Fig.6</A> : Effects of BDNF (50 ng/ml), S100<SUB><IMG SRC="../images/beta.gif"
ALT="beta"></SUB> (10 ng/ml), BDNF(50 ng/ml) + S100<SUB><IMG SRC="../images/beta.gif"
ALT="beta"></SUB> (10 ng/ml), BDNF (50 ng/ml) + anti S100<SUB><IMG SRC="../images/beta.gif"
ALT="beta"></SUB> (1/10,000 dilution), and BDNF (50 ng/ml) + IgG (1/10,000, dilution) on the soma area
of 5-HT-IR cells.</STRONG> The cells were treated for 3 days, at which time they were fixed and subjected
to 5-HT immunocytochemistry. The mean values obtained from 3 independent experiments were calculated.
The results were analyzed using ANOVA followed by tukey honest test. Data were presented as the mean +
SEM. *: statistically significant (p<0.05) in comparison to the control value; **: statistically significant (p<0.05)
compared to the BDNF treated group.
<P>
<A NAME="fig.7"></A><P>
<A HREF="ano3fo7l.html"> <IMG SRC="../images/an03f07s.gif" ALT="Fig.7" Border=0><br clear=both>
<STRONG>Fig.7</A> : Effects of BDNF (50 ng/ml), S100<SUB><IMG SRC="../images/beta.gif"
ALT="beta"></SUB> (10 ng/ml), BDNF(50 ng/ml) + S100<SUB><IMG SRC="../images/beta.gif"
ALT="beta"></SUB> (10 ng/ml), and BDNF (50 ng/ml) + anti S100<SUB><IMG SRC="../images/beta.gif"
ALT="beta"></SUB> (1/10,000 dilution) on the total length of 5-HT-IR cells.</STRONG> The cells were
treated for 3 days, at which time they were fixed and subjected to 5-HT immunocytochemistry. The mean
values obtained from 3 independent experiments were calculated. The results were analyzed using ANOVA
followed by tukey honest test. Data were presented as the mean + SEM. *: statistically significant (p<0.05) in
comparison to the control value.
<P>
<A NAME="fig.8"></A><P>
<A HREF="ano3fo8l.html"> <IMG SRC="../images/an03f08s.jpg" ALT="Fig.8" Border=0><br clear=both>
<STRONG>Fig.8</A>: A speculative diagram of the dynamic instability model suggesting possible trophic
interactions between BDNF and S100<SUB><IMG SRC="../images/beta.gif"
ALT="beta"></SUB>.</STRONG> The model of neuronal dynamic instability is based on the spontaneous
depolymerization studies of microtubules (Michison and Kirschner, 1984). Our diagram shows BDNF effects
on neurons and glial cells (Blue line). It can be seen that in neurons BDNF results in the stimulation of
Ca<sup>2+</sup> and kinase activity which would favor a phosphorylation of MAP-2, tau and GAP-43. The
absence of these proteins would encourage the spontaneous depolymerization of microtubules and
microfilaments (Red line). In glial cells, the increase in Ca<sup>2+</sup> and kinase (Blue line) could promote
a production and release of S100<SUB><IMG SRC="../images/beta.gif" ALT="beta"></SUB> (Green Line).
BDNF also promotes the synthesis and release of 5-HT which would also release S100<SUB><IMG
SRC="../images/beta.gif" ALT="beta"></SUB> through the 5-HT-1A receptor (Green Line).
S100<SUB><IMG SRC="../images/beta.gif" ALT="beta"></SUB> by inhibiting the action of PKC prevents
the phosphorylation of MAP-2, tau and GAP-43. These proteins can stabilize microtubules and microfilaments
(Green Line). The immediate actions of BDNF would be to promote depolymerization and branching, although
long-term exposure may result in cytoskeletal collapse. On the other hand, S100<SUB><IMG
SRC="../images/beta.gif" ALT="beta"></SUB> promotes polymerization and results in a stabilization and
extension of processes.
<P>
<HR>
<A NAME="acknowledge"></A>
<FONT SIZE=+2><STRONG>Acknowledgments</STRONG></FONT><P>
We are grateful to Dr. L. Mamounas for helpful discussion during the course of this work. We would like to
thank Xia Ping Hou for excellent technical assistance. This work has been supported by the funds from NIA
program project 1 P01 AG10208.<P>
<HR>
<A NAME="references"></A>
<FONT SIZE=+2><STRONG>References</STRONG></FONT><P>
Aoki, C., Siekevitz, P. (1988) Pasticity in brain development. Scientific American 259: 56-64.
<P>
Azmitia, E.C., Dolan, K., Whitaker-Azmitia, P.M.(1990) S100<SUB><IMG SRC="../images/beta.gif"
ALT="beta"></SUB> but not NGF, EGF, insulin or calmodulin is a CNS serotonergic growth factor. Brain
Res. 516: 534-536.
<P>
Azmitia, E.C., Hou, X.P. (1994) Steroid regulation of neurotrophic activity: Primary microcultures of midbrain
raphe and hippocampus. In: Methods in Neuroscience 22; Neurobiology of steroids. DeKloet, E.R. and
Sutanto, W., eds. Academic Press, San Diego, CA, pp359-371.
<P>
Azmitia, E.C., Rubinstein, V.J., Strafaci, J.A., Rios, J.C., and Whitaker-Azmitia, P.M. (1995) 5-HT1A agonist
and dexamethasone revarsal of parachloroamphetamine induced loss of MAP2 and synaptophysin
immunoreactivity in adult rat brain. Brain Res. 677:181-192.
<P>
Barbacid, M. (1994) The trk family of neurotrophin receptors. J. Neurobiology, 25: 1386-1403.
<P>
Baudier, J., Mochly-Rosen, D., Newton, A. (1987) Comparison of S100<SUB><IMG
SRC="../images/beta.gif" ALT="beta"></SUB> protein with calmodulin: interactions with melittin and
microtubule-associated tau proteins and inhibition of phosphorylation of tau proteins by PKC. Biochem. J. 25:
2886-2893.
<P>
Baudier, J., Cole, R.D. (1988) Interactions between the microtubule-associated tau proteins and
S100<SUB><IMG SRC="../images/beta.gif" ALT="beta"></SUB> regulate tau phosphorylation by the
Ca<sup>2+</sup>/Calmodulin-dependent protein kinase II. J. Biol. Chem. 263: 5876-5882.
<P>
Diez-Guerra, F.J., Avila, J. (1993) MAP2 phosphorylation parallels dendrite arborization in hippocampal
neurons in culture. Neuroreport 4: 419-422.
<P>
Eaton, M. J., Staley, J.K., Globus, M. Y.-T., Whittemore, S.R. (1995) Developmental regulation of early
serotonergic neuronal differentiation: the role of brain-derived neurotrophic factor and membrane depolarization.
Dev. Biol. 170: 169-182.
<P>
Frisen, J., Verge, V.M.K., Fried, K., Risling, M., Person, H., Trotter, J., Hokfelt, T., Lindholm, D. (1993)
Charactrization of glial trkB receptors: differential response to injury in the central and peripheral nervous
systems. Proc. Natl. Acad. Sci. 90: 4971-4975.
<P>
Haring, J.H., Hagan, A., Olson, J., Rodgers, B. (1993) Hippocampal serotonin levels influence the expression
of S100<SUB><IMG SRC="../images/beta.gif" ALT="beta"></SUB> detected by immunocytochemistry.
Brain Res. 631:119-123.
<P>
Hesketh, J., Baudier, J. (1986) Evidence that S100 proteins regulate microtubule assembly and stability in rat
brain extracts. Int. J. Biochem. 18: 691-695.
<P>
Klein, R., Nanduri, V., Jing, S., Lamballe, F., Tapley, P., Bryant, S., Cordon-Cardo, C., Jones, K.R.,
Reichardt, L.F., Barbacid, M. (1991) The trkB tyrosine protein kinase is a receptor for brain-derived
neurotrophic factor and neurotrophin-3. Cell, 66: 395-403.
<P>
Kligman, D., Marshak, D.R. (1985) Purification and characterization of neurite extension factor from bovine
brain. Proc. Natl. Acad. Sci. 82:7136-7139.
<P>
Liu, J.P. and Lauder, J.M. (1992) S100<SUB><IMG SRC="../images/beta.gif" ALT="beta"></SUB> and
insulin-like growth factor-II differentially regulate growth of developing serotonin and dopamine neurons in vitro.
J. Neurosci. Res. 33: 248-256.
<P>
Mamounas, L.A., Blue, M.E., Siuciak, J.A., Altar, C.A. (1995) Brain-derived neurotrophic factor promotes
the survival and sprouting of serotonergic axons in rat brain. J. Neurosci. 16: 7929-7939.
<P>
Marsh, H.N., Scholz, W.K., Lamballe, F., Klein, R., Nanduri, V., Barbacid, M., Palfrey, H.V. (1993) Signal
transduction events mediated by the BDNF receptor gp145trkB in primary hippocampal pyramidal cell culture.
J. Neurosci. 13: 4281-4292.
<P>
Merlio, J-P., Ernfors, P., Jaber, M., Persson, H. (1992) Molecular cloning of rat trkC and distribution of cells
expressing messenger RNAs for members of the trk family in the rat central nervous system. Neurosci. 51:
513-532.
<P>
Mitchison, T.M., Kirschner, M.W. (1984) Dynamic instability of microtubule growth. Nature 312: 237-242.
<P>
Nishi, M., Whitaker-Azmitia, P.M., Azmitia, E.C. (1996) Enhanced synaptophysin immunoreactivity in rat
hippocampal culture by 5-HT1A agonist, S100<SUB><IMG SRC="../images/beta.gif" ALT="beta"></SUB>,
and corticosteroid receptor agonists. SYNAPSE 23: 1-9.
<P>
Nishida, E., Hoshi, M., Miyata, Y., Sakai, H., Kadowaki, T., Kasuga, M., Saijo, S., Ogawara, H., Akiyama,
T. (1987) Tyrosine phosphorylation by the epidermal growth factor receptor kinase induces functional
alterations in microtubule-associated protein 2. J. Biol. Chem. 262:16200-16204
<P>
Roback, J.D., Marsh, H.N., Downen, M., Palfrey, H.C., Wainer, B. H. (1995) BDNF-activated signal
transduction in rat cortical glia. Europ. J. Neurosci. 7:849-863.
<P>
Sheu, F-S, Azmitia, E.C., Marshak, D.R., Parker, P.J. (1994) Glial-derived S100<SUB><IMG
SRC="../images/beta.gif" ALT="beta"></SUB> protein selectively inhibits recombinant protein kinase C
(PKC) phosphorylation of neuron-specific protein F1/GAP43. Mol. Brain Res. 21:62-66.
<P>
Ueda, S., Kokotos-Leonardi, E.T., Bell III, J., Azmitia, E.C. (1995) Serotonergic sprouting into transplanted
C-6 gliomas is blocked by S100<SUB><IMG SRC="../images/beta.gif" ALT="beta"></SUB> antisense
gene. Mol. Brain Res. 29: 365-368.
<P>
VanEldik, L.J., Christie-Pope, B., Bolin, L.M., Shooter, E.M., Whetsell, Jr., W.O. (1991) Neurotrophic
activity of S100<SUB><IMG SRC="../images/beta.gif" ALT="beta"></SUB> in cultures of dorsal root
ganglia from embryonic chick and fetal rat. Brain Res. 542: 280-285.
<P>
White, L.A., Eaton, M. J., Castro, M.C., Klose, K.J., Globus, M. Y.-T., Shaw, G., Whittemore, S.R. (1994)
Distinct regulatory pathways control neurofilament expression and neurotransmitter synthesis in immortalized
serotonergic neurons. J. Neurosci.14: 6744-6753
<P>
Whitaker-Azmitia, P.M., Murphy, R., Azmitia, E.C. (1990) Stimulation of astroglial 5-HT1A receptors releases
the serotonergic growth factor, protein S100, and alters astroglial morphology. Brain Res. 528: 155-158.
<P>
Whittemore, S.R., White, L.A. (1993) The regulation of neuronal differentiation in a temperature-sensitive cell
line derived from medullary raphe. Brain Res. 615: 27-40.
<P>
WidmerH.R., Knusel, B., Hefti, F. (1992) Stimulation of phosphatidylinositol hydrolysis by brain-derived
neurotrophic factor and neurotrophin-3 in rat cerebral cortical neurons developing in culture. J. Neurochem. 59:
2113-2124.
<P>
WidmerH.R., Kaplan, D.R., Rabin S.J., Beck, K.D., Hefti, F., Knusel, B (1993) Rapid phosphorylation of
phospholipase C <IMG SRC="../images/gamma.gif" ALT="gamma">1 by Brain-derived neurotrophic factor
and neurotrophin-3 in cultures of embryonic rat cortical neurons. J. Neurochem. 60: 2111-2123.
<P>
Zhou, F.C., Azmitia, E.C., Bledsoe, S. (1995) Rapid serotonergic fiber sprouting in response to ibotenic acid
lesion in the striatum and hippocampus. Brain Res. Dev. Brain Res. 84: 89-98.
<P>
<hr>
<center>
<table>
<tr>
<td align=center><a href="index.html" TARGET="_top"><img src="../images/b_back.gif" border=0
ALT="Back"></a>
<td align=left><a href="#top"><img src="../images/b_top.gif" border=0 ALT="Top"></a>
<tr>
<td><a href="../../articles.html" TARGET="_top"><img src="../images/b_ita1.gif" border=0 ALT="Index to
Articles"></a>
<td><a href="../../index.html" TARGET="_top"><img src="../images/b_home1.gif" border=0 ALT="Return
Home"></a>
</table><p>
</center>
]]>