help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rage, F.
Right arrow Articles by Tapia-Arancibia, L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rage, F.
Right arrow Articles by Tapia-Arancibia, L.
Right arrowPubmed/NCBI databases
*Compound via MeSH
*Substance via MeSH
Endocrinology Vol. 140, No. 2 909-916
Copyright © 1999 by The Endocrine Society

ARTICLES

Brain-Derived Neurotrophic Factor and Neurotrophin-3 Enhance Somatostatin Gene Expression through a Likely Direct Effect on Hypothalamic Somatostatin Neurons1

Florence Rage, Béatrice Riteau, Gérard Alonso and Lucia Tapia-Arancibia

Laboratoire de Plasticité Cérébrale (F.R., B.R., L.T.-A.), EP 628 CNRS and U336 INSERM (G.A.), Université de Montpellier 2, 34095 Montpellier, Cedex 5, France

Address all correspondence and requests for reprints to: Dr. Lucia Tapia-Arancibia, Laboratoire de Plasticité Cérébrale, EP 628 CNRS, Université de Montpellier 2, Place Eugène Bataillon, 34095 Montpellier Cédex 5, France. E-mail: aranci{at}crit.univ-montp2.fr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Although neurotrophins (NTs) have been extensively studied as neuronal survival factors in some areas of the central nervous system, little is known about their function or cellular targets in the hypothalamus. To understand their functional significance and sites of action on hypothalamic neurons, we examined the effects of their cognate ligands on neuropeptide content and messenger RNA (mRNA) expression in somatostatin neurons present in fetal rat hypothalamic cultures. Treatments were performed in defined insulin-free medium between days 6 and 8 of culture, since the maximal effects of NTs on somatostatin content and mRNA expression were observed after 48-h incubations. Brain-derived neurotrophic factor and NT-3, but not nerve growth factor, induced a dose-dependent increase in somatostatin content, which was influenced by plating density. The same treatment increased somatostatin mRNA and immunostaining intensity of somatostatin neurons, but had no effect on the number of these labeled neurons. The increased levels of somatostatin (peptide and mRNA) induced by NTs were not blocked by tetrodotoxin or by glutamate receptor antagonists, suggesting that endogenous neurotransmitters (e.g. glutamate) were not involved in these effects. In contrast, the stimulatory effects were completely blocked by K-252a, an inhibitor of tyrosine kinase (Trk) receptors, whereas the less active analog K-252b was ineffective. Double-labeling studies demonstrated that both TrkB or TrkC receptors were located on somatostatin neurons. Our results show that, in rat hypothalamic cultures, brain-derived neurotrophic factor, and NT-3 have a potent stimulatory effect on peptide synthesis in somatostatinergic neurons, likely through direct activation of TrkB and TrkC receptors.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE NEUROTROPHIN (NT) family, a group of structurally related neurotrophic factors, all expressed in neurons of the central nervous system, includes nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), NT-3, NT-4/5, and NT-6 (1, 2, 3, 4, 5, 6). Their biological effects are mediated by high-affinity tyrosine kinase (Trk) receptors, although all bind to a low-affinity receptor (p75) (7). NGF binds to TrkA receptors, BDNF preferentially activates Trk B receptors, whereas NT-3 mainly interacts with TrkC receptors but also stimulates TrkB receptors. Trk receptor activation initiates differentiation and survival processes in selected neuronal populations (8).

Within the hypothalamus, moderate levels of BDNF, low levels of NGF, and no NT-3 messenger RNA (mRNA) have been localized in adult or newborn rats (9, 10, 11, 12, 13, 14, 15). It is noteworthy that, besides the hippocampus, the hypothalamus is the region where the highest levels of BDNF protein are found in adult rat brain (16, 17). In situ hybridization studies have shown that, in adult rats, TrkB and TrkC mRNA are expressed in most cells of all hypothalamic nuclei, whereas TrkA mRNA is rarely detected in the hypothalamus (13, 18). In RT-PCR studies, we detected the expression of the three high-affinity receptor mRNAs in adult hypothalamus (15). Despite this huge body of morphologic data, suggesting an important autocrine or paracrine role in the hypothalamus, little is known (19, 20) about the function or target neurons of NTs in this structure.

As a first step toward understanding the putative role of NTs in the hypothalamus, we examined the long-term effects of NTs on somatostatin synthesis, because NTs increase the expression of some neuropeptides in other structures of the central nervous system (21). We thus examined the effects of Trk receptor activation by their cognate ligands (NGF, BDNF, or NT-3), in terms of their possible involvement in regulating somatostatin expression (peptide content or mRNA accumulation) in primary cultures of hypothalamic neurons. We also investigated whether the effects observed were direct or meditated by endogenous neurotransmitters.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Hypothalamic cell cultures
Primary cultures were prepared by mechanoenzymatic dissociation of fetal (day 17) Sprague Dawley rat hypothalami, as previously described (22), with necessary modifications. Briefly, cells were plated in 35-, 22-, or 16-mm plastic culture dishes (Falcon, Lincoln Park, NJ) for immunocytochemical analysis or to measure RNA or somatostatin content, respectively. Dishes were previously coated with poly-D-lysine (10 µg/ml; 220,000 mol wt) and preincubated for 1 h with 10% FCS in MEM (Grand Island Biological Co., Grand Island, NY). Unless indicated, cells were seeded in growth medium composed of MEM supplemented with 10% Nu serum (Collaborative Research, Lexinton, MA), insulin (5 µg/ml), glucose (0.6%), glutamine (2 mM), penicillin-streptomycin (2.5 U/ml), adjusted to pH 7.4 with 5 mM HEPES. Cultures were maintained at 37 C in a humid atmosphere (95% air-5% CO2). The proliferation of nonneuronal cells was inhibited by treatment with 10 µM cytosine-arabinoside for 48 h between days 4 and 6 after plating. After 6 days culture, the growth medium was replaced by a defined serum-free medium. This insulin-and NT-free medium was essentially that described by Bottenstein and Sato (23) and adjusted to pH 7.4 with 5 mM HEPES. For time-course studies, cells were plated and grown in defined serum-free medium. For all other treatments, after changing the medium to serum-free conditions, cultures were exposed for 48 h to the different concentrations of either BDNF (generously provided by Regeneron Pharmaceuticals, Inc., Tarrytown, NY), or NGF and NT-3 (generously provided by Genentech, Inc., San Francisco, CA). Experiments were performed after 8 days culture (DIV), because we previously showed that somatostatin mRNA levels are stabilized at this point (24). In our previous studies, we also observed that the amounts of somatostatin secreted in the medium were always approximately 1% of those contained in the neurons. The cellular somatostatin content can be taken as a reliable criterion for somatostatin production.

Somatostatin RIA
Cellular somatostatin content was determined in evaporated aliquots (20 µl) of cell homogenates extracted from individual dishes in 1 M acetic acid. Immunoreactive somatostatin was determined by a sensitive RIA (22), carried out in the same tube to minimize peptide loss. Binding curves were run in tubes containing 20 µl of evaporated 1N acetic acid. The assay has a sensitivity of 1 pg/ml, and the antiserum (N° 2044), which was used at 1:70,000 dilution, was generously provided by the Institut Pasteur (Paris, France). The intra- and interassay coefficients of variation were 6.1% (n = 15) and 7.4% (n = 12), respectively.

RNA extraction from primary cultures
Total RNA was extracted from hypothalamic neuronal cultures using the procedure of Peppel and Baglioni (25), optimized for RNA extraction from cultured cells.

Antisense digoxigenin-labeled RNA probes
Steady-state levels of somatostatin mRNA were quantitated by Northern blot analysis of cellular RNA using an antisense nonradioactive digoxigenin-labeled complementary RNA (cRNA).

A somatostatin probe was constructed by inserting a fragment of rat preprosomatostatin cDNA into the expression vector pSP65 (26). The vector containing the somatostatin cDNA insert was linearized with SalI and a digoxigenin-labeled antisense RNA probe transcribed with an SP6 polymerase.

A 28S ribosomal RNA (rRNA)-specific riboprobe was used as internal standard to establish the specificity of treatments and the relative amounts of RNA in each sample. This probe was transcribed from a cDNA template (pTRI-RNA-28S) purchased from Ambion, Inc. (Austin, TX) and consisted of a 115-bp human 28S rRNA gene fragment. The linearized plasmid was transcribed using T7 RNA-polymerase.

Digoxigenin-labeled antisense somatostatin and 28S rRNA probes were prepared using the DIG RNA-labeling kit, according to the manufacturer’s instructions (Boehringer Mannheim, Meylan, France), with a minor modification (addition of 30 mM dithiotreitol) to improve labeling efficiency. RNA accumulation was expressed as mean ± SEM of arbitrary densitometry units. Ratios between somatostatin mRNA vs. 28S rRNA hybrydization were calculated to estimate the effects of each treatment, which were then expressed as percentages of control ratios obtained in the absence of NT treatment.

Northern blot analysis
Ten micrograms of total RNA was electrophoresed in 1.1% denaturing agarose gel (6.6% formaldehyde, 1 x 4-morpholino-propane-sulfonic acid), and capillary blotted on nylon membrane (Biodyne A; 0.2 µm, Pall, Portsmouth, UK) at room temperature with 10 x SSC. After RNA blotting, the membrane was baked at 120 C for 30 min to fix the RNA. Prehybridization was carried out at 68 C for 1 h using a solution containing 50% formamide, 5 x SSC, 2% blocking reagent (Boehringer Mannheim), 0.1% N-lauryl sarkosine, and 0.02% SDS. Hybridization was performed at 68 C for 16 h in the same buffer containing the denatured digoxigenin-cRNA probes, 100 ng/ml for somatostatin and 15 ng/ml for 28S rRNA. Membranes were washed twice for 5 min in 2 x SSC and 0.1% SDS at room temperature and twice for 15 min at 68 C in the same washing buffer. The last washing was carried out under stringent conditions, i.e. at 68 C in 0.1 x SSC and 0.1% SDS for 25 min.

Chemiluminescent signal detection was performed using the CDP-Star detection kit according to the manufacturer’s instructions (Boehringer Mannheim). After exposure, Kodak X-O-Mat AR films (NEN Life Science Products, Inc., Boston, MA) were analyzed by quantification using Sigma Chemical Co. gel software (Jandel Scientific Software GmbH, Erkrath, Germany).

Immunocytochemical procedures
Single immunostaining. The procedure used was described previously (27). Cultures were fixed by 1-h incubation in 4% paraformaldehide in 0.1 M phosphate buffer at pH 7.4. After careful rinsing in PBS, they were treated for single staining of somatostatin. The antiserum used was a rabbit IgG polyclonal antibody against somatostatin (N° 2044, kindly provided by the Institut Pasteur) at 1:2000 dilution in PBS containing 0.1% Triton X-100, 1% BSA, and 1% normal goat serum. After 12-h incubation with the primary antibody, cultures were rinsed in PBS and incubated for 2 h in a corresponding secondary antibody conjugated to fluorescein (goat antirabbit IgG antibody, Sigma Chemical Co.), 1:200 dilution. Immunostained cultures were observed and photographed under a Zeiss axioskop fluorescence microscope in 24 nonadjacent fields per culture, and the number of immunostained neurons was counted. The histochemical specificity of the antibody was confirmed by the complete disappearance of immunostaining when cultures were incubated without primary antibody or with the primary antibody previously adsorbed with synthetic somatostatin (50 µg/ml diluted antibody).

Double immunostaining. After being fixed as described above, cultures were incubated with two primary antibodies, including: 1) a rat IgG monoclonal antibody against somatostatin (Chemicon, Temecula, CA, 1:50 dilution); and 2) one of the two rabbit IgG polyclonal antibodies against TrkB and TrkC (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, diluted 1:200). After 12-h incubation with primary antibodies, cultures were rinsed in PBS and incubated for 2 h with two secondary antibodies against rat and mouse IgG coupled to fluorescein and Cy3, respectively. Primary and secondary antibodies were diluted, as described above, in PBS containing 0.1% Triton X-100, 1% BSA, and 1% normal goat serum. Double-immunostained cultures were observed under a fluorescence microscope equipped with the appropriate fluorescence filters.

Statistical analysis
The results were analyzed with a one-way ANOVA, followed by Barlett’s test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Time-course study
To address the question of whether NTs stimulate somatostatin expression, we examined the effects of 16-, 24-, or 48-h exposure of hypothalamic neurons (density: 2 x 105 cells/cm2) to 30 ng/ml of NGF, BDNF, or NT-3 on peptide content. Figure 1AGo shows that maximal peptide increase was observed after 48-h exposure to BDNF or NT-3. To determine whether the NT-induced peptide augmentation was correlated with increased somatostatin mRNA, we measured the cellular accumulation of mRNA under similar experimental conditions. Figure 1BGo shows that the stimulatory effect of BDNF on somatostatin mRNA levels had a similar pattern to that obtained for somatostatin content. Moreover, when BDNF was added to the incubation medium for 6 or 8 days, somatostatin immunoreactivity or mRNA levels were not significantly different from those measured after 2-day treatments. NT-3 presented the same kinetic patterns, whereas NGF had no effect at any time or concentration used (results not shown). Thereafter, the time selected to study the effects of NTs on somatostatin expression was 48 h between days 6 and 8 in vitro.



View larger version (32K):
[in this window]
[in a new window]
  		Figure 1. Time-course study of BDNF and NT-3 on somatostatin synthesis. A, NTs (30 ng/ml) were added 16, 24, and 48 h before peptide extraction, which was performed at day 8 of culture; B, neurons were exposed to BDNF (30 ng/ml) during 2 (6 DIV), 6 (2 DIV) and 8 days (plating day) before peptide or mRNA extraction in a defined, serum-free, insulin- and NT-free medium. Steady-state somatostatin mRNA levels were quantitated by Northern blot analysis of cellular RNA using an antisense nonradioactive digoxigenin-labeled cRNA. A digoxigenin-labeled 28S rRNA-specific riboprobe was used as internal standard. The chemiluminescent signal was detected using the CDP-Star detection kit, and RNA accumulation was expressed as arbitrary densitometry units. The results are presented as ratios of somatostatin mRNA to 28S rRNA hybrydization, which are then expressed as percentages of control ratios obtained in the absence of NT treatment. After peptide extraction in 1 M acetic acid, aliquots (20 µl) were evaporated and assessed for somatostatin content, by RIA. The results are means ± SEM of three determinations from three independent experiments. *, P < 0.05; **, P < 0.01 vs. control.

 
Dose-response effect of BDNF and NT-3 on somatostatin content and influence of cell plating density
Under the indicated experimental conditions (i.e. neuronal density 2 x 105 cells/cm2), when cultures were treated with 30, 70, or 140 ng/ml of NTs, the magnitude of the response was not dose-dependent (results not shown). One explanation was that possible endogenous cellular production of NTs could have masked the effect of the exogenous addition; or, secondly, a maximal effect might have already been reached at the lowest concentration studied. According to the former hypothesis, a decrease in the plating density of the cultures would thus lead to better NT stimulation. Consequently, cultures prepared with plating densities of 2, 1.5, 1, or 0.5 x 105 cells/cm2 were exposed for 48 h to 70 ng/ml of NTs. Interestingly, the maximal response elicited by BDNF or NT-3 was obtained at a density of 1 x 105 cells/cm2 (Fig. 2Go). In agreement with the first hypothesis formulated above, when cells were plated at a density of 1 x 105 cells/cm2, BDNF and NT-3, within the 5–140 ng/ml range, induced dose-dependent responses with maximal effects at 70 ng/ml of BDNF (Fig. 3bGo) or NT-3 (Table 1Go). The stimulatory effect of BDNF on somatostatin content was significantly higher than that induced by NT-3. Under the same experimental conditions, BDNF and NT-3 significantly (P < 0.01) stimulated somatostatin mRNA accumulation (Fig. 3aGo and Table 1Go), whereas NGF was ineffective in stimulating the peptide content (Fig. 2Go) or mRNA levels (Fig. 4Go). BDNF presented a slightly higher efficacy than NT-3 in stimulating somatostatin mRNA. At the lowest plating density used (0.5 x 105 cells/cm2), NTs had no effect either on intracellular peptide content or on mRNA levels. Thereafter, the cell density selected was 1 x 105 cells/cm2.



View larger version (46K):
[in this window]
[in a new window]
  		Figure 2. Effect of plating density on the stimulatory effects of BDNF and NT-3 on somatostatin content. Cells were plated at densities of 0.5, 1, 1.5, or 2 x 105/cm2 and, after 6 days of in vitro culture, exposed to 70 ng/ml BDNF, NT-3, or NGF for 48 h in a defined, insulin-free medium. After peptide extraction in 1 M acetic acid, aliquots (20 µl) were evaporated and assessed for somatostatin content, by RIA. Data are expressed as pg somatostatin/dish. Values indicate means ± SEM of three independent experiments. Some bars are not visible because of the very low SEM. **, P < 0.01 vs. corresponding control value.

 


View larger version (37K):
[in this window]
[in a new window]
  		Figure 3. Dose-response effect of BDNF on somatostatin mRNA and on somatostatin content. Hypothalamic neurons were cultured for 6 days in vitro and then exposed to the indicated concentrations of BDNF for 48 h in a defined, insulin-free medium. Somatostatin mRNA was quantitated by Northern blot analysis, and somatostatin content was assessed by RIA, as indicated in Fig. 1Go. Controls are equal to 100%, and the data are expressed as a percentage of control levels. Values indicate means ± SEM of three independent experiments performed in triplicate. *, P < 0.05; **, P < 0.01 vs. control.

 

View this table:
[in this window]
[in a new window]
  		Table 1. Effect of NT-3 on somatostatin mRNA and somatostatin content

 


View larger version (44K):
[in this window]
[in a new window]
  		Figure 4. Northern blot analysis of somatostatin mRNA in control (C) and in BDNF-, NT-3-, and NGF-treated neurons. After 6 days in vitro, cultures were exposed for 48 h to 70 ng/ml of BDNF, NT-3, and NGF in a defined insulin-free medium. Total RNA was isolated as described in Materials and Methods. Ten micrograms of total RNA was electrophoresed per lane. Nonradioactive, DIG-labeled cRNA was used to visualize the somatostatin mRNA, and a DIG-labeled antisense fragment (115 bp) was used to visualize the 28S RNA used as internal control. The histogram shows the statistical representation of ratios of somatostatin vs. 28S RNA hybridization. The results are expressed as a percentage of control levels. Controls are equal to 100%. *, P < 0.05 vs. control; **, P < 0.01 vs. control.

 
Increased somatostatin turnover by BDNF and NT-3 treatment
We attempted to gain insight into the mode by which NTs increased somatostatin expression. Three possible situations could be proposed: first, NTs may promote the survival of somatostatinergic neurons; second, NTs may stimulate somatostatin turnover and thus increase somatostatin mRNA and peptide content in somatostatinergic neurons; and third, NTs may change the phenotype of silent neurons that could start synthesizing the peptide after treatment. To test these hypotheses, control and NT-treated neurons were immunocytochemically stained with a specific antibody against somatostatin and then counted. Table 2Go shows that the number of somatostatin-positive neurons did not change after NT treatment. However, qualitative staining differences were observed, by light microscopy, between treated and untreated (70 ng/ml) cultures (Fig. 5Go). In control cultures (Fig. 5AGo), fibers were rarely labeled; and in immunostained perikarya, labeling was essentially associated with punctiform perinuclear structures, probably corresponding to the Golgi apparatus, which has been shown to contain high somatostatin concentrations (28). In contrast, the whole perikarya and all fibers were intensely labeled in numerous BDNF- or NT-3-treated neurons (Fig. 5Go, B and C).


View this table:
[in this window]
[in a new window]
  		Table 2. Effect of BDNF and NT-3 on the number of somatostatin neurons

 


View larger version (78K):
[in this window]
[in a new window]
  		Figure 5. Immunofluorescence detection of somatostatin in control (A) and in BDNF- (B) and NT-3- (C) treated cultures. After 6 days of in vitro culture, cells were treated for 48 h without (A) or with 70 ng/ml BDNF (B) or 70 ng/ml NT-3 (C) in a defined, insulin-free medium. In control conditions (A), somatostatin immunostaining was essentially associated with dot-like structures and with scarce axonal profiles (arrow in A). After 48 h exposure to BDNF (B) or to NT-3 (C) treatments, more intense immunostaining seemed to be associated with the whole cytoplasm of a number of perikarya and with numerous varicose axonal profiles (arrows in A and B). A–C, x400; bar, 50 µm.

 
Trk involvement in NT-induced somatostatin expression
To investigate potential involvement of Trk receptors in the stimulatory effects of BDNF and NT-3, described here, we inhibited Trk activities with K252a or K252b, two structurally related Trk receptor inhibitors (29, 30). Cultures were treated for 48 h with the inhibitors alone or added at the same time as the NTs. K252a (100 nM) completely blocked the stimulatory effects of BDNF (Fig. 6aGo) and NT-3, whereas K252b (1 µM) had no effect on the BDNF-induced mRNA accumulation (Fig. 6bGo). K252b added alone did not significantly influence somatostatin mRNA content, whereas K252a had a weak (but significant) inhibitory effect.



View larger version (35K):
[in this window]
[in a new window]
  		Figure 6. Effect of the protein Trk inhibitors K252a and K252b on NT-induced somatostatin mRNA accumulation. a, At 6 days of culture, 100 nM of K-252a was added to the culture medium alone or along with BDNF or NT-3. A control group was tested in standard conditions, i.e. with incubation medium alone, in the absence of NTs or the inhibitor K252a. b, K252b, a weaker Trk inhibitor, was ineffective in blocking BDNF-induced somatostatin mRNA accumulation. Total mRNA was prepared as indicated in Materials and Methods. Each lane contained 10 µg RNA. DIG-labeled cRNAs were used to visualize somatostatin and 28S RNA, respectively. Controls are equal to 100%, and data are expressed as a percentage of control levels. Values indicate means ± SEM of two independent experiments. *, P < 0.05; a, P < 0.05, b, P < 0.01 vs. control.

 
Direct effect of NTs on somatostatin neurons
Three approaches were used to study whether the stimulatory effect of NTs on somatostatin gene expression was direct or secondary to the action of NTs on other neurons present in the cell cultures: 1) The effects of BDNF and NT-3 were examined in the presence of tetrodotoxin (TTX, a specific voltage-dependent sodium channel blocker) to reduce possible indirect effects induced via nearby neuronal depolarization; 2) The effects of glutamate receptor antagonists on BDNF-induced effects were studied; and 3) We looked for the presence of NT receptors on somatostatin neurons. Neither TTX (1 µM) (Fig. 7Go) nor a broad range of glutamate receptor blockers modified somatostatin mRNA accumulation induced by BDNF (Fig. 8Go) or NT-3 (results not shown). Indeed, when the glutamate receptor antagonists MCPG (a metabotropic receptor antagonist), DNQX or MK-801 (two ionotropic receptor antagonists), tested at 1 mM, 100 µM, and 10 µM concentrations, respectively, were applied simultaneously with BDNF or with NT-3, somatostatin mRNA levels were the same as those measured in the presence of NTs alone. Double immunohistochemical staining of cultured hypothalamic neurons revealed that both TrkB (Fig. 9Go, A and A’) and TrkC (Fig. 9Go, B and B’) receptors were located on somatostatinergic neurons.



View larger version (62K):
[in this window]
[in a new window]
  		Figure 7. Lack of effect of TTX on mRNA levels enhanced by BDNF. Between days 6 and 8 of culture, cells were grown in medium containing 1 µM TTX or 1 µM TTX + BDNF (70 ng/ml). A control group was tested in standard conditions, i.e. in incubation medium alone, in the absence of NTs or TTX (C). TTX treatment did not modify somatostatin mRNA levels induced by BDNF. Results are means of 4–5 samples from two independent experiments and are expressed relative to untreated control, which was considered as 100%. **, P < 0.01 vs. control.

 


View larger version (51K):
[in this window]
[in a new window]
  		Figure 8. Glutamate receptor antagonists did not modify BDNF-induced somatostatin mRNA expression. The glutamate receptor antagonists MCPG (1 mM), DNQX (100 µM), or MK-801 (10 µM) were added to the defined insulin-free culture medium between days 6 and 8 in vitro, alone or along with BDNF. Somatostatin mRNA levels were unchanged, compared with levels measured in the presence of BDNF alone. Results are means ± SEM from three independent experiments (n is indicated in parenthesis) and are expressed relative to untreated control, which was considered as 100%. *, P < 0.05; **, P < 0.01 vs. control.

 


View larger version (98K):
[in this window]
[in a new window]
  		Figure 9. Double fluorescence immunostaining of cultures for somatostatin and TrkB (A and A') or for somatostatin and TrkC (B and B'). The large majority of neuronal cell bodies immunostained for somatostatin (A and B) also exhibits immunostaining for TrkB (arrows in A and A') or for TrkC (arrows in B and B'). Note that immunostainings for TrkB (A') or TrkC (B') are associated with a number of cell bodies that are somatostatin-negative. Bar, 50 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have demonstrated that BDNF and NT-3 have a potent and specific stimulatory effect on hypothalamic somatostatinergic neurons. In our hands, BDNF was more efficient than NT-3 in stimulating somatostatin content. Considering the high levels of BDNF in the adult rat hypothalamus (17) and the high expression of BDNF mRNA in the periventricular nucleus of adult rat hypothalamus (15), where hypophysiotropic somatostatin is synthesized, our results provide serious evidence of a physiological role of this NT in this structure. Indeed, we observed that a 48-h treatment of cultures with BDNF or NT-3 significantly increased cellular somatostatin content and steady-state somatostatin mRNA levels in primary cultures of hypothalamic neurons. The specificity of this effect was highlighted by the lack of any increase in somatostatin content in NGF-treated cultures. Furthermore, these data are in line with the results of previous studies, where NGF was shown to have no effect on peptidergic neurons (31). Examination of somatostatin-immunopositive neurons, after BDNF or NT-3 treatment, indicated no change in the number of somatostatin neurons; but the immunoreactivity in each somatostatin neuron was intensified, suggesting that the somatostatin level was increased in each neuron by NTs. NTs thus seemed to stimulate somatostatin turnover. Our data were consistent with previous reports conducted on fetal mesencephalic or whole brain neurons, indicating no change in the overall cell number of dopaminergic (32) or NPY (31) neurons after BDNF treatment. Our present observations thus indicated that stimulation of somatostatin production was the primary effect of BDNF, rather than promoting the survival of somatostatin neurons, at least at this developmental period.

At present, we do not know whether NTs act at transcriptional or posttranscriptional levels. A better understanding of the mode of action of NTs on peptide biosynthesis would require further studies, including analysis of mRNA stability or investigation of possible effects on transcription, through nuclear run-on experiments. NT activation of Trk receptors involves multiple second-messenger pathways that may underlie these effects. Recent studies indicated that the transcription factor cAMP response element-binding protein (CREB) is an important regulator of BDNF-induced gene expression in cortical neurons and hippocampal slices (33). On the other hand, we previously showed that cAMP increases somatostatin biosynthesis, in experiments conducted in a similar paradigm, with fetal rat hypothalamic cells, or in clonal isolates of NIH-3T3 fibroblast cells transfected with the rat somatostatin gene (34). CREB may therefore be one of the signaling pathways by which BDNF activates somatostatin gene expression in hypothalamic neurons. In agreement with this hypothesis, Montminy et al. (35) demonstrated the presence of a cAMP-responsive element within the rat somatostatin gene.

NT effects in the hippocampus, brain cortex, or other extrahypothalamic regions in the central nervous system have been thoroughly studied (36, 37, 38). For instance, previous studies showed that NTs enhance the development of septal cholinergic neurons (39), hippocampal neurons (40), striatal GABAergic neurons (41), and mesencephalic dopaminergic neurons (32, 42). However, in the hypothalamus (where they might play a major role during the development of neuroendocrine neurons) or in the adult animal, in different physiological situations, very few data are available (12, 14, 20).

In vivo administration of BDNF in newborn rat brain increases the contents of neuropeptide Y, substance P, somatostatin, enkephalin and cholecystokinin in brain cortex, striatum, and hippocampus (21). Intraventricular injection of BDNF linearly increased the expression of somatostatin mRNA in the anterior neocortex, reaching maximum levels at 48 h, whereas the same doses of NGF had no influence on neuropeptide levels (21). In contrast, these authors failed to detect any marked influence of BDNF on somatostatin or neuropeptide Y expression in the hypothalamus. In addition to the major fact that completely different methods were used (in vivo vs. in vitro methods), the differences, relative to our results, could be explained in at least three ways. Nawa et al. (21) found a relatively restricted area of action of BDNF, close to the cannula route implanted into the lateral ventricle. Consequently, diffusion of BDNF to the third ventricle could be limited, and no significant amounts of BDNF would be delivered to the hypothalamus. A second explanation is that, at birth, peptidergic neurons may have reached their maturity and lost their plasticity, with respect to neuropeptide expression, whereas this plasticity may still be possible in fetal hypothalamic neurons. This possibility is made less likely by the observation that infusions of BDNF in the cortex of adult rat produced elevations of somatostatin peptide and somatostatin mRNA (43). The third and most likely explanation is that, at neonatal stages, endogenous NT levels are sufficient to ensure peptidergic expression, and exogenous administration of NTs would therefore not further alter the peptide phenotype. In the present study, using appropriate densities of fetal dissociated neurons, we demonstrated a positive regulatory role of NTs on hypothalamic neurons secreting somatostatin. In this way, NTs could even amplify their trophic action, because somatostatin itself was also reported to be a trophic factor in PC12 cells (44).

The present data provide strong evidence that the effect of NTs on somatostatinergic neurons is direct and is not dependent upon the release of other endogenous substances released by the cultures. In fact, TTX, a specific voltage-dependent sodium channel blocker, as well as a broad range of glutamate receptor antagonists, did not prevent BDNF or NT-3-mediated induction of somatostatin. Glutamate might have been a good candidate, in view of previous data showing that BDNF increases K+-evoked glutamate release in cultured cortical neurons (45). NGF is also able to induce a rapid increase in both spontaneous and K+-evoked glutamate release in synaptosomes from adult rat hippocampus (46) or in hippocampal embryonic neurons (47). Because we have shown that glutamate stimulates somatostatin synthesis through NMDA-type receptors (27), we examined its possible mediation in the stimulatory effect of NTs. In addition, very recent data show that BDNF, NT-3, and NGF modulate NMDA receptors, through the glycine site in cultured hippocampal and striatal mouse neurons (48), suggesting another possibility of glutamate-mediated mechanisms. The present pharmacologic experiments clearly indicated that glutamate is not involved in the stimulatory effects of NTs in hypothalamic somatostatinergic neurons. Further evidence of a direct effect of NTs was obtained in double immunocytochemical staining experiments, revealing that TrkB and TrkC receptors were located on somatostatinergic neurons. The activation of Trk receptors by BDNF and NT-3 was confirmed by experiments using K-252a, a potent Trk inhibitor. In our hands, K252b did not inhibit BDNF-induced somatostatin mRNA expression. In agreement with our results, in rat hippocampal slices, K252b was ineffective in blocking BDNF-mediated synaptic responses (49). In the present work, we did not look for the presence of the low-affinity P75NTR receptor; but its involvement is unlikely, because NGF did not affect the biological responses measured here. This is further supported by the fact that K252a blocks the two parameters analyzed, somatostatin content and somatostatin mRNA. However, we cannot totally rule out the potential role of other endogenous neurotransmitters released through a TTX-insensitive mechanism in the BDNF- and NT-3-induced stimulations.

In conclusion, our data represent the first evidence of a specific effect of NTs on a peptidergic neuronal population at the hypothalamic level. These results provide evidence that NTs, at least in part, directly regulate somatostatin gene expression. This study highlights the fact that cultured hypothalamic neurons, secreting somatostatin, express functional receptors responding to BDNF and NT-3 and could thus be considered as a choice model to investigate action mechanisms involved in the long-term effects of NTs.


   Acknowledgments
 
We wish to thank Dr. Franz Hefti and members of Genentech, Inc. for the generous gift of recombinant human (h)NGF and recombinant hNT-3, Dr.George D. Yancopoulos and members of Regeneron Pharmaceuticals, Inc. for the generous gift of hBDNF, and Dr. Stephanie Lee for the rat somatostatin cDNA clone. We also thank Dr. Sandor Arancibia for critical reading of the manuscript and Mr. Edmond Savary for technical assistance.


   Footnotes
 
1 This work was supported by training grants (to F.R.) from the Société de Secours des Amis des Sciences de l’Institut de France (1996) and Fondation IPSEN (1997). Back

Received June 23, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Leibrock J, Lottspeich F, Hohn A, Hofer M, Hengerer B, Masiakowski P, Thoenen H, Barde YA 1989 Molecular cloning and expression of brain-derived neurotrophic factor. Nature 341:149–152[CrossRef][Medline]
  2. Maisonpierre PC, Belluscio L, Squinto S, Ip NY, Furth ME, Lindsay RM, Yancopoulos GD 1990 Neurotrophin-3: a neurotrophic factor related to NGF and BDNF. Science 247:1446–1451[Abstract/Free Full Text]
  3. Hohn A, Leibrock J, Bailey K, Barde YA 1990 Identification and characterization of a novel member of the nerve growth factor/brain-derived neurotrophic factor family. Nature 344:339–341[CrossRef][Medline]
  4. Berkemeier LR, Winslow JW, Kaplan DR, Nikolics K, Goeddel DV, Rosenthal A 1991 Neurotrophin-5: a novel neurotrophic factor that activates trkA and trkB. Neuron 7:857–866[CrossRef][Medline]
  5. Ip NY, Ibáñez CF, Nye SH, McClain J, Jones PM, Gies DR, Belluscio L, Le Beau MM, Espinosa III R, Squinto SP, Persson H, Yancopoulos GD 1992 Mammalian neurotrophin-4: structure, chromosomal localization, tissue distribution, and receptor specificity. Proc Natl Acad Sci USA 89:3060–3064[Abstract/Free Full Text]
  6. Götz R, Köster R, Winkler C, Raulf F, Lottspeich F, Schartl M, Thoenen H 1994 Neurotrophin-6 is a new member of the nerve growth factor family. Nature 372:266–269[CrossRef][Medline]
  7. Chao MV, Hempstead BL 1995 p75 and Trk: a two-receptor system. Trends Neurosci 18:321–326[CrossRef][Medline]
  8. Chao MV 1992 Neurotrophin receptors: a window into neuronal differentiation. Neuron 9:583–593[CrossRef][Medline]
  9. Korsching S, Auburger G, Heumann R, Scott J, Thoenen H 1985 Levels of nerve growth factor and its mRNA in the central nervous system of the rat correlate with cholinergic innervation. EMBO J 4:1389–1393[Medline]
  10. Ernfors P, Wetmore C, Olson L, Persson H 1990 Identification of cells in rat brain and peripheral tissues expressing mRNA for members of the nerve growth factor family. Neuron 5:511–526[CrossRef][Medline]
  11. Friedman WJ, Olson L, Persson H 1991 Cells that expressed brain-derived neurotrophic factor mRNA in the developing postnatal rat brain. Eur J Neurosci 3:688–697[CrossRef][Medline]
  12. Castrén E, Thoenen H, Lindholm D 1995 Brain-derived neurotrophic factor messenger RNA is expressed in the septum, hypothalamus and in adrenergic brain stem nuclei of adult rat brain and is increased by osmotic stimulation in the paraventricular nucleus. Neuroscience 64:71–80[CrossRef][Medline]
  13. Ojeda SR, Hill DF, Katz KH 1991 The genes encoding nerve growth factor and its receptor are expressed in the developing female rat hypothalamus. Mol Brain Res 9:47–55[Medline]
  14. Smith MA, Makino S, Kim SY, Kvetnansky R 1995 Stress increases brain-derived neurotrophic factor messenger ribonucleic acid in the hypothalamus and pituitary. Endocrinology 136:3743–3750[Abstract]
  15. Marmigère F, Rage F, Tapia-Arancibia L, Arancibia S 1998 Expression of mRNAs encoding BDNF and its receptor in adult rat hypothalamus. Neuroreport 6:1959–1963
  16. Nawa H, Carnahan J, Gall C 1995 BDNF protein measured by a novel enzyme immunoassay in normal brain and after seizure: partial disagreement with mRNA levels. Eur J Neurosci 7:1527–1535[CrossRef][Medline]
  17. Katoh-Semba R, Takeuchi IK, Semba R, Kato K 1997 Distribution of brain-derived neurotrophic factors in rats and its changes with development in the brain. J Neurochem 69:34–42[Medline]
  18. Merlio JP, 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. Neuroscience 51:513–532[CrossRef][Medline]
  19. Ojeda SR, Dissen GA, Junier MP 1992 Neurotrophic factors and female sexual development. Front Neuroendocrinol 13:120–162[Medline]
  20. Berg-von der Emde K, Dees WL, Hiney JK, Hill DF, Dissen GA, Costa ME, Moholt-Siebert M, Ojeda SR 1995 Neurotrophins and the neuroendocrine brain: different neurotrophins sustain anatomically and functionally segregated subsets of hypothalamic dopaminergic neurons. J Neurosci 15:4223–4237[Abstract]
  21. Nawa H, Pelleymounter MA, Carnahan J 1994 Intraventricular administration of BDNF increases neuropeptide expression in newborn rat brain. J Neurosci 14:3751–3765[Abstract]
  22. Tapia-Arancibia L, Astier H 1988 Glutamate stimulates somatostatin release from diencephalic neurons in primary culture. Endocrinology 123:2360–2366[Abstract/Free Full Text]
  23. Bottenstein JE, Sato GH 1979 Growth of neuroblastoma cell line in serum-free supplemented medium. Proc Natl Acad Sci USA 76:514–517[Abstract/Free Full Text]
  24. Rage F, Rougeot C, Tapia-Arancibia L 1994 GABAA and NMDA receptor activation controls somatostatin messenger RNA expression in primary cultures of hypothalamic neurons. Neuroendocrinology 60:470–476[Medline]
  25. Peppel K, Baglioni C 1990 A simple and fast method to extract RNA from tissue culture cells. Biotechniques 9:711–713[Medline]
  26. Goodman RH, Aron DC, Roos BA 1983 Rat preprosomatostatin: structure and processing by microsomal membranes. J Biol Chem 258:5570–5573[Abstract/Free Full Text]
  27. Rage F, Alonso G, Tapia-Arancibia L 1993 Stimulatory effect of N-methyl-D-aspartate on somatostatin gene expression in cultured hypothalamic neurons. Mol Brain Res 17:287–294[Medline]
  28. Alonso G 1988 Effects of colchicine on the intraneuronal transport of secretory material prior to the axon: a morphofunctional study in hypothalamic neurosecretory neurons of the rat. Brain Res 453:191–203[CrossRef][Medline]
  29. Koizumi S, Contreras ML, Matsuda Y, Hama T, Lazarovici P, Guroff G 1988 K-252a: a specific inhibitor of the action of nerve growth factor on PC12 cells. J Neurosci 8:715–721[Abstract]
  30. Knüsel B, Hefti F 1991 K-252b is a selective and nontoxic inhibitor of nerve growth factor action on cultured brain neurons. J Neurochem 57:955–962[CrossRef][Medline]
  31. Barnea A, Cho G, Lu G, Mathis M 1995 Brain-derived neurotrophic factor induces functional expression and phenotypic differentiation of cultured fetal neuropeptide Y-producing neurons. J Neurosci Res 42:638–647[CrossRef][Medline]
  32. Hyman C, Hofer M, Barde YA, Juhasz M, Yancopoulos GD, Squinto SP, Lindsay RM 1991 BDNF is a neurotrophic factor for dopaminergic neurons of the substantia nigra. Nature 350:230–232[CrossRef][Medline]
  33. Finkbeiner S, Tavazoie SF, Maloratsky A, Jacobs KM, Harris KM, Greenberg ME 1997 CREB: a major mediator of neuronal neurotrophin responses. Neuron 19:1031–1047[CrossRef][Medline]
  34. Montminy MR, Low MJ, Wu J, Tapia-Arancibia L, Reichlin S, Goodman RH 1986 Somatostatin gene is regulated by cyclic AMP. J Neurosci 6:1171–1186[Abstract]
  35. Montminy MR, Sevarino KA, Mandel JA, Goodman RH 1986 Identification of a cyclic-AMP-responsive element within the rat somatostatin gene. Proc Natl Acad Sci USA 83:6682–6686[Abstract/Free Full Text]
  36. Glass DJ, Yancopoulos GD 1993 The neurotrophins and their receptors. Trends Cell Biol 3:262–268
  37. Lindsay RM 1994 Neurotrophins and receptors. Prog Brain Res 103:3–14[Medline]
  38. Thoenen H 1995 Neurotrophins and neuronal plasticity. Science 270:593–598[Abstract/Free Full Text]
  39. Hefti F 1986 Nerve growth factor promotes survival of septal cholinergic neurons after fimbrial transections. J Neurosci 6:2155–2162[Abstract]
  40. Ohsawa F, Widmer HR, Knusel B, Denton TL, Hefti F 1993 Response of embryonic rat hippocampal neurons in culture to neurotrophin-3, brain-derived neurotrophic factor and basic fibroblast growth factor. Neuroscience 57:67–77[CrossRef][Medline]
  41. Mizuno K, Carnahan J, Nawa H 1994 Brain-derived neurotrophic factor promotes differentiation of striatal GABAergic neurons. Dev Biol 165:243–256[CrossRef][Medline]
  42. Knüsel B, Winslow JW, Rosenthal A, Burton LE, Seid DP, Nikolics K, Hefti F 1991 Promotion of central cholinergic and dopaminergic neuron differentiation by brain-derived neurotrophic factor but not neurotrophin-3. Proc Natl Acad Sci USA 88:961–965[Abstract/Free Full Text]
  43. Croll SD, Wiegand SJ, Anderson KD, Lindsay RM, Nawa H 1994 Regulation of neuropeptides in adult rat forebrain by the neurotrophins BDNF and NGF. Eur J Neurosci 6:1343–1353[CrossRef][Medline]
  44. Ferriero DM, Sheldon RA, Messing RO 1994 Somatostatin enhances nerve growth factor-induced neurite outgrowth in PC12 cells. Dev Brain Res 80:13–18[CrossRef][Medline]
  45. Takei N, Sasaoka K, Inoue K, Takahashi M, Endo Y, Hatanaka H 1997 Brain-derived neurotrophic factor increases the stimulation-evoked release of glutamate and the levels of exocytosis-associated proteins in cultured cortical neurons from embryonic rats. J Neurochem 68:370–375[Medline]
  46. Knipper M, Leung LS, Zhao D, Rylett RJ 1994 Short-term modulation of glutamatergic synapses in adult rat hippocampus by NGF. Neuroreport 5:2433–2436[Medline]
  47. Leßmann V, Gottmann K, Heumann R 1994 BDNF and NT-4/5 enhance glutamatergic synaptic transmission in cultured hippocampal neurones. Neuroreport 6:21–25[Medline]
  48. Jarvis CR, Xiong Z-G, Plant JR, Churchil D, Lu W-Y, MacVicar BA, MacDonald JF 1997 Neurotrophin modulation of NMDA receptors in cultured murine and isolated rat neurons. J Neurophysiol 78:2363–2371[Abstract/Free Full Text]
  49. Tanaka T, Saito H, Matsuki N 1997 Inhibition of GABAA synaptic responses by brain-derived neurotrophic factor (BDNF) in rat hippocampus. J Neurosci 17:2959–2966[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
K. Khoufache, F. LeBouder, E. Morello, F. Laurent, S. Riffault, P. Andrade-Gordon, S. Boullier, P. Rousset, N. Vergnolle, and B. Riteau
Protective Role for Protease-Activated Receptor-2 against Influenza Virus Pathogenesis via an IFN-{gamma}-Dependent Pathway
J. Immunol., June 15, 2009; 182(12): 7795 - 7802.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
N. Cougot, S. N. Bhattacharyya, L. Tapia-Arancibia, R. Bordonne, W. Filipowicz, E. Bertrand, and F. Rage
Dendrites of Mammalian Neurons Contain Specialized P-Body-Like Structures That Respond to Neuronal Activation
J. Neurosci., December 17, 2008; 28(51): 13793 - 13804.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
N. Piazzon, F. Rage, F. Schlotter, H. Moine, C. Branlant, and S. Massenet
In Vitro and in Cellulo Evidences for Association of the Survival of Motor Neuron Complex with the Fragile X Mental Retardation Protein
J. Biol. Chem., February 29, 2008; 283(9): 5598 - 5610.
[Abstract] [Full Text] [PDF]

Home page
 J EndocrinolHome page
L. Givalois, G. Naert, L. Tapia-Arancibia, and S. Arancibia
Involvement of brain-derived neurotrophic factor in the regulation of hypothalamic somatostatin in vivo.
J. Endocrinol., March 1, 2006; 188(3): 425 - 433.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rage, F.
Right arrow Articles by Tapia-Arancibia, L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rage, F.
Right arrow Articles by Tapia-Arancibia, L.
Right arrowPubmed/NCBI databases
*Compound via MeSH
*Substance via MeSH

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals