ABSTRACT
(Neuroscience-Net, Volume 1, Article #10003; June
9, 1996)
Neurotrophic factors have effects on both the survival and the differentiation of neurons. The majority of these factors are produced by glial cells. However, neurons synthesize brain-derived neurotrophic factor (BDNF), a potent neurotrophic member of NGF family. BDNF is known to exert a multitude of trophic effects on diverse neuronal populations, including central serotonergic and dopaminergic neurons through trkB receptor. In serotonergic neurons or RN46A cells, BDNF increases survival and serotonin synthesis. When BDNF is slowly administered directly in the adult, serotonergic-depleted cerebral cortex of the rat, serotonergic fibers in the region show a dramatic sprouting response and an increase in 5-HT levels and uptake. Surprisingly, the increased innervation persists for months after BDNF infusion is terminated. This long-term action may involve activation of the trophic activity of glial cells. Glial cells, following stimulation of the 5-HT-1A receptor, release S100ß which stimulates the extension of serotonergic and cortical fibers. Recently, it has been shown that cultured rat cortical glial cells respond to BDNF although they contain predominantly truncated trkB receptors. Are some of the trophic effects exerted by BDNF mediated via S100ß?
The effects of BDNF and S100ß on serotonergic properties were investigated in fetal rat raphe primary cultures grown in serum-free condition without serotonin or steroids. While BDNF (50 ng/ml) promoted the number of serotonin (5-HT) immunoreactive (IR) neurons (+78%), S100ß (10 ng/ml) application did not produce an increase in neuronal number. BDNF stimulated morphological differentiation by increasing the soma area of 5-HT-IR neurons (+35%) without increasing neurite extension. In contrast, S100ß promoted the extension of 5-HT-IR neurites (+110%), but did not increase their soma area. BDNF treatment also enhanced the S100ß immunoreactivity in glial cells. A combined treatment with BDNF (50 ng/ml) and S100ß (10 ng/ml) increased the 5-HT-IR cell numbers (+67%) and the soma area (+34%), but did not increase the neurites length. An antibody to S100ß, but not normal IgG, blocked the increase in soma area and the increase in survival of 5-HT-IR neurons induced by BDNF. These results suggest that BDNF and S100ß are active protein growth factors on serotonergic neurons and BDNF may require trophic interactions with S100ß to achieve its full neurotrophic potential.
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 S100ß (Azmitia et al., 1990; Liu and Lauder, 1992) and BDNF (Eaton et al., 1995; Mamounas et al.,1995; White et al.,1994). S100ß, 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 S100ß from glial cells is induced by 5-HT1A agonists (Whitaker-Azmitia et al., 1990).
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).
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 S100ß, we investigated the effects of BDNF on S100ß immunoreactivity, effects of BDNF and S100ß on serotonergic properties, and the actions of anti- S100ß antibody on these BDNF effects in primary raphe cultures.
Primary Culture
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 CO2, 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% Na2HPO4.12H2O, 0.003% KH2PO4, 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 8x105 cells/cm2 by adding 200 ul of the cell suspension to each well (area of 0.28 cm2; 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 CO2 incubator at 37 oC with 5% CO2/95% air.
Growth factor treatment
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 mg/ml bovine serum albumine (BSA)). At this time point, cultures were supplemented with human recombinant BDNF (50 ng/ml, Promega), S100ß(10 ng/ml, 99% homogeneous by SDS-PAGE, East Acres Biologicals, MA), BDNF (50 ng/ml) + S100ß (10 ng/ml) or BDNF (50 ng/ml) + rabbit anti-S100ß antibody (1/10,000 dilution of purified IgG fraction, a gift from Dr. L. VanEldik). BDNF and S100ß 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.
Immunocytochemistry
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 S100ß (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% H2O2 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.
Morphometric Analysis
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. S100ß -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).
Statistical analysis
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.
Descriptive analysis of BDNF and S100ß on rat brainstem raphe cultures
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 S100ß positive glial cells after 4 days of culture (data not shown). The effects of BDNF (50ng/ml) or S100ß (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 S100ß immunoreactivity was analyzed. Figures 1 (low magnification) and 2 (high magnification) show 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 (Figs 1C and 2C) and BDNF+S100ß (Figs 1D and 2D), 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 S100ß (Figs 1B and >2B), 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.
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BDNF promotes S100ß immunoreactivity in glial cells
In order to investigate whether exogenous BDNF directly affects S100ß level in glial cells, S100ß immunoreactivity was examined in raphe cultures treated with BDNF for 3 h or 2 days. In BDNF-treated cultures, S100ß immnuoreactivity was stimulated after 3h (Fig. 3) and 2 days (data not shown) of treatment. Particularly, in 3h BDNF-treated cultures, S100ß immunoreactivity was increased within and around the astrocytes. In BDNF-treated glial cells, there was an increased appearance of membrane blebbing showing S100ß-IR. Quantitative image analysis indicated that BDNF produced a 53 % increase (p<0.05) in S100ß immunoreactivity as compared to control values and also shifted the distribution of S100ß-IR upward (Fig.4). The increase in S100ß seen in raphe cultures was also observed in hippocampal cultures which contained no exogenous or intrinsic source of 5-HT (data not shown).
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Survival of 5-HT-IR neurons increased by BDNF but not S100ß in raphe cultures
As shown in Figure 5, BDNF treatment and combining BDNF treatment with S100ß for 3 days significantly increased the number of 5-HT-IR cells (+78% and +68% of control value, respectively, p<0.05), whereas S100ß did not show a significant increase. Total numbers of viable cells determined by trypan-blue in each well treated with BDNF, S100ß, a combination of BDNF and anti-S100ß 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).
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Morphological differentiation of 5-HT-IR neurons induced by BDNF or S100ß
The effects of BDNF or S100ß 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 S100ß did not show a significant increase in the soma area (Fig.6). 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 (Fig. 7). In contrast, S100ß significantly promoted the total process length (+110%, P<0.01), especially, the length of the longest process. As observed in Fig.2B, S100ß treatment increased the number of 5-HT-IR processes. Combined treatment of BDNF and S100ß induced an increase in the soma area (+34%, p<0.05) but not in the total process length (+20%, not significant).
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Anti-S100ß antibody blocks BDNF trophic-actions on 5-HT-IR neurons
The question of possible trophic interactions between BDNF and S100ß was addressed by using anti-S100ß antibody blocking method. Combining BDNF treatment with anti-S100ß antibody showed a decrease in the number of 5-HT-IR cells as compared to BDNF treated cultures (-30%, p<0.05) (Figs. 2E and 5). Also an antibody to S100ß blocked the effect of BDNF on the soma area (-24% as compared to BDNF treatment, p<0.05) (Figs. 1E,2E, and 6). In contrast, anti-S100ß 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.
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 S100ß in primary raphe cultures.
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.
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 
1 (Widmer
et al., 1992,1993), which result in c-fos induction and Ca2+
-mobilization. These signal transduction events may contribute
to the serotonergic neuronal survival. In contrast to BDNF, exogenously
added S100ß did not affect the survival of 5-HT-IR
cells in primary raphe culture.
Immunocytochemical studies showed that BDNF treatment for both 3 h and 2 days increased the S100ß level in glial cells. Especially, in the 3h treatment cultures, S100ß-IR was more intense inside and around the glial cells. The pattern has been observed in vivo and may indicate released S100ß (Zhou et al, 1995). The astrocytes after BDNF treatment show S100ß in blebbs evaginating from the plasma membrane. These results suggest that BDNF promotes the process of producing and/or releasing of S100ß. 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 S100ß 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 S100ß from astrocytes is induced by 5-HT1A agonists (Whitaker-Azmitia et al., 1990). In the adult, removal of 5-HT induces a reduction in S100ß (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 S100ß do not require neuronal support (Zhou et al., 1995). The current studies are examining if the BDNF-induced increase in S100ß 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 S100ß immunoreactivity in hippocampal glial cells (unpublished data).
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 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, S100ß 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). S100ß 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. S100ß 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 S100ß plays an active role in serotonergic
sprouting. The C6 glioma cell line, that secretes S100ß,
transplanted into an adult rat hippocampus induces the sprouting
of serotonergic fibers (Ueda et al., 1995).
The question of possible trophic interactions between BDNF and S100ß was addressed by using an antibody blocking approach. An antibody to S100ß, 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 S100ß which is stimulated in raphe cultures treated with BDNF, although an exogenously added S100ß has no effect on the survival. An antibody to S100ß 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 S100ß which stabilizes cytoskeletal proteins such as MAP2 and tau, but also by promoting kinase activities which might be essential for the initialization of arborization and/or sprouting of neurites. Together with the other results of the present study, this antibody blocking approach supported trophic interactions between BDNF and S100ß.
BDNF stimulated the morphological differentiation by increasing the soma area of 5-HT-IR neurons without increasing the neurite extension. S100ß 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 S100ß. 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 S100ß, 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 S100ß but not result in process elongation, while S100ß applied alone does produce process elongation. In fact, when we applied both BDNF and S100ß, there was no process outgrowth. As shown in the diagram (Fig.8), S100ß 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 S100ß. Further studies are needed to explore this interaction.
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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.
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.
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