regulation of brain-derived neurotrophic factor transcripts by neuronal activation in rat...

Regulation of Brain-Derived NeurotrophicFactor Transcripts by Neuronal Activation inRat Hypothalamic Neurons

Frederic Marmigere, Florence Rage, and Lucia Tapia-Arancibia*Laboratoire de Plasticite Cerebrale, Universite Montpellier 2, Montpellier, France

Brain-derived neurotrophic factor (BDNF) belongs to theneurotrophin family and regulates the survival, differen-tiation, and maintenance of function in different neuronalpopulations. BDNF is strongly expressed in hypotha-lamic neurons, where it exerts long- or short-lasting ac-tions. Because glutamate has been associated with reg-ulations of hypothalamic hormones, we examined theregulation of the four promoters of the BDNF gene byglutamate in fetal hypothalamic neurons. The expressionlevels of BDNF transcripts were investigated using semi-quantitative RT-PCR. BDNF protein was determined byenzyme immunoassay, and BDNF and Trk B (BDNF re-ceptor) gene variations were determined by RNAse pro-tection assay. By RT-PCR, we showed that, under basalconditions, BDNF transcripts from exons I, II, and III butnot from IV were expressed in the hypothalamic neurons.Glutamate increased expression of both the protein andthe four transcripts via N-methyl-D-aspartate receptors,with maximal stimulations after 3 hr of application forexon I and II mRNAs and after 1 hr for exon III and IVmRNAs. Actinomycin D blocked the increase of all tran-scripts, whereas cycloheximide treatment inhibited stim-ulation only of exon I and II mRNAs. Trk B mRNA wasrapidly and transiently reduced after glutamate applica-tion. Our results demonstrate that glutamate 1) regulatesBDNF mRNA expression at an early developmental stagein hypothalamic neurons and 2) exerts a differential reg-ulation of BDNF transcripts. J. Neurosci. Res. 66:377–389, 2001. © 2001 Wiley-Liss, Inc.

Key words: BDNF transcripts; promoters; NMDA; hypo-thalamic neurons; Trk B

Alternative splicing of mRNA is a mode of physio-logical regulation that also occurs in many genes of theendocrine system. Hormones and other metabolic signalsmay regulate this phenomenon (Chew, 1997). This mech-anism combined with the existence of tissue-specific al-ternative promoters provides multiple possible regulationsfor complex genes whose final translation products areinvolved in various actions. Alternative promoters arecommonly used to express the same gene product indifferent cell types (Shapiro et al., 1991). Members of theneurotrophin family, a group of structurally related trophic

factors expressed in neurons of the central nervous system(Lewin and Barde, 1996), including endocrine neurons,provide an example of these types of regulation. Forinstance, neurotrophin-3 (NT-3) and brain-derived neu-rotrophic factor (BDNF) genes give rise to different tran-scripts (Timmusk et al., 1993; Kendall et al., 2000).

The rat BDNF gene has a complex structure, withfour, short, 59 noncoding exons (exons I–IV) containingseparate promoters and one 39 exon (exon V) encodingthe mature BDNF protein (see Fig. 3A). Alternative usageof these promoters and differential splicing result in fourBDNF mRNAs with different 59 unstranslated exons andthe same coding exon. In addition, each transcription unituses two different polyadenylation signals in the 39 end ofexon V that altogether generate eight distinct transcripts.The biological significance of the eight distinct BDNFmRNAs is unknown, although interestingly each BDNFmRNA encodes an identical BDNF protein. Much efforthas been made to investigate the regulation of BDNFmRNA expression using exon V-specific probes that labelthe totality of BDNF transcripts. However, less informa-tion is available on the regulation of individual expressionpatterns of the different transcripts. The existence of mul-tiple promoters seems to determine the tissue-specific ex-pression of the rat BDNF gene (Timmusk et al., 1993;Bishop et al., 1994). Thus, it has been shown that thesetranscripts are differentially expressed across brain regions(Timmusk et al., 1993; Bishop et al., 1994). mRNAscontaining exons I, II, and III are predominantly expressedin the brain, whereas promoter IV is more active inperipheral tissues, e.g., lung and heart.

Together with the hippocampus, the hypothalamusis the brain region containing the highest BDNF proteinlevels (Nawa et al., 1995; Katoh-Semba et al., 1997). Inaddition, the mRNA (Merlio et al., 1992) encoding itshigh-affinity tyrosine kinase-coupled receptor (Trk B) aswell as Trk B immunostaining (Yan et al., 1997) are foundin most hypothalamic nuclei. For hypothalamic neurons,

*Correspondence to: Dr. L. Tapia-Arancibia, Laboratoire de PlasticiteCerebrale, UMR 5102 CNRS, Universite Montpellier 2, CC 090 PlaceEugene Bataillon, 34095 Montpellier Cedex 5, France.E-mail: [email protected]

Received 12 March 2001; Revised 5 May 2001; Accepted 18 June 2001

Journal of Neuroscience Research 66:377–389 (2001)

© 2001 Wiley-Liss, Inc.

we and others very recently described short-lasting (Mar-migere et al., 2001) and long-lasting (Rage et al., 1999;Loudes et al., 1999, 2000) biological effects of BDNF,indicating that hypothalamic neurons are highly respon-sive to BDNF. In vivo, BDNF seems to be associated withstress responses in the hypothalamus (Smith et al., 1995;Castren et al., 1995).

In the CNS, BDNF expression is substantially in-creased by manipulations stimulating neuronal activity(Lindholm et al., 1994; Lindvall et al., 1994). Glutamaterelease could trigger this increase in hypothalamic neuronsinasmuch as it has been shown that systemic injections ofkainic acid stimulate BDNF expression in hippocampus orcerebral cortical neurons in vivo (Metsis et al., 1993;Falkenberg et al., 1996). In addition, glutamate is animportant neurotransmitter increased in the hypothalamusafter stress exposure (Palkovits et al., 1985) and is involvedin the control of hypothalamic hormones also regulated byBDNF (Tapia-Arancibia and Astier, 1988; Marmigere etal., 2001).

In this study, we have used exon-specific probesfrom the rat BDNF gene to demonstrate a differentialactivation by glutamate of the four BDNF promoters inthese neurons. We also investigated the effect of glutamateon the expression of BDNF and Trk B genes and onBDNF protein variation. Glutamate receptors involved inthese regulations were pharmacologically characterized.Finally, we determined whether transcription or proteinsynthesis inhibition had selective effects on the distinctBDNF transcripts.

MATERIALS AND METHODS

Hypothalamic Cell Cultures

Primary cultures were prepared by mechanoenzymaticdissociation of fetal (day 17) Sprague-Dawley rat hypothalami aspreviously described (Rage et al., 1999). Briefly, before plating,dishes were precoated with poly-D-lysine (10 mg/ml; 220,000molecular weight) and preincubated for 1 hr with 10% fetal calfserum in minimum essential medium (MEM; GIBCO, GrandIsland, NY). After withdrawal of the last coating solution, cellswere seeded at a density of 7.5 3 104 cells/cm2 in growthmedium composed of MEM supplemented with 10% Nu serum(Collaborative Research, Lexinton, MA), insulin (5 mg/ml),glucose (0.6%), glutamine (2 mM), and penicillin-streptomycin(2.5 U/ml), adjusted to pH 7.4 with 5 mM HEPES. Cultureswere maintained at 37°C in a humid atmosphere (95% air/5%CO2). Nonneuronal cell proliferation was inhibited by a 48 hrtreatment with 10 mM cytosine arabinoside between days 4 and6 after plating. After 6 days of culture, the growth medium wasreplaced by normal medium. Experiments were performed afterday 10 of culture.

RNA Extraction From Culture Tissues

Total RNA was extracted from primary hypothalamic cellcultures using Peppel and Baglioni’s (1990) method. RNAconcentration and purity were evaluated by spectrometry byoptical density (OD) measurements at 260 and 280 nm.

Semiquantitative RT-PCR Assay

For detection of the different BDNF transcripts, RT wascarried out for 1 hr at 42°C using 200 U of Super Script II RT(Gibco BRL Life Technologies, Cergy Pontoise, France). Be-cause the exon III mRNA is abundant in the hypothalamicneurons, aliquots contained 1 mg of total cellular RNA in 20 mlfor BDNF mRNA containing exons I, II, and IV or 100 ng oftotal cellular RNA for BDNF mRNA containing exon III. Thereaction also contained 4 ml of 53 first-strand buffer (GibcoBRL), 25 pmol oligo-(dT) primer (Roche, Meylan, France),0.5 mM of each dNTP nucleotide (Promega, Charbonnieres,France), and 10 mM dithiotreitol (Gibco BRL).

The 59 and 39 sequences used for exons I, II, III, and IVwere previously described by Bishop et al. (1994), with slightpreviously described modifications (Marmigere et al., 1998).The antisense and sense sequences used for cyclophilin are59-CATGCCTTCTTTCA-CCTTCCCAAAGAC-39 and 59-CGTGCTCTGAGCACTGGGGAGAAA-39, respectively. Theycorrespond to nt 450–424 and 151–174, respectively, in the ratcyclophilin mRNA sequence. All parameters (percentage of RTproducts, primer quantity, magnesium concentration, and num-ber of cycles) were determined as recommended (Ma et al.,1994), so that signals obtained for BDNF exons and cyclophilinwere not saturated.

PCR was then carried out in a two-part 50 ml finalreaction volume. Part A contained 2 ml of RT reaction mixture,5 ml of 103 PCR buffer (Promega), 0.5 mM of each dNTPnucleotide (Promega), 2.5 mM MgCl2 (Promega), 25 pmol ofPCR sense primer for BDNF exons (Genosys, Cambridge,England), and 2 pmol of sense primer for cyclophilin (Genosys).After we overlaid cocktail A with a drop of mineral oil, themixture was held in a thermal cycler (Bioblock, Illkirch, France)at 94°C for 3 min to inactivate the RT. Thereafter, cocktail Bcontaining 25 pmol of PCR antisense primer for BDNF exons(Genosys), 2 pmol of antisense primer for cyclophilin (Genosys),and 2.5 U of Taq DNA polymerase (Promega) was added toeach sample through the oil. This procedure avoided dimerformation and further amplification. cDNA was then amplifiedfor 31 cycles for BDNF mRNA containing exons I, II, and IVor 32 cycles for transcripts containing exon III. Each cycleincluded 1 min of denaturation at 95°C, 1 min of annealing at54°C, and 1.5 min of extension at 72°C, except for the lastcycle, which was followed by a 15 min extension at 72°C.

Amplified cDNA was then separated by electrophoresis in2% agarose gels stained with ethydium bromide. Photos of thegel were analyzed using the NIH Image software (version 1.62non-PFU; NIH, Bethesda, MD). BDNF exon signals werenormalized with respect to those of cyclophilin. The ratio isexpressed as percentage of control 6SEM.

RT-PCR products were cloned and sequenced by “Ge-nome Express” (Grenoble, France). This analysis showed thatthey corresponded to the sequences of exons I, II, III, and IV inthe rat cDNA.

Probe Preparation

Cloning of exon V BDNF PCR product. RT reac-tion was conducted as described below using 1 mg of total RNAextract. Exon V BDNF cDNA was amplified using 2 ml of the

378 Marmigere et al.

RT mixture for 30 cycles as previously described. The 59 and 39primer sequences used for exon V BDNF are 59-GGAGCTGAGCGTGTGTGACAGTA-39 and 59-CCATG-GGATTACACTTGGTCCTCG-39 and correspond to nt 486–508 and 646–624, respectively, in the exon V sequence codingfor the preproprotein. PCR products were separated by elec-trophoresis in a 3% low-melting-point agarose gel (Bioprobe,Montreuil, France) stained with ethydium bromide. The am-plified sequence corresponding to the expected 186 bp size wasexcised, and cDNA was then electroeluted using 0.75-inch-diameter dialysis tubing (Life Technologies). cDNA was thencloned within the pGEM-T vector according to the manufac-turer instructions (Promega). Linearization with Sal I (Promega)and T7 (Promega) polymerase-directed transcription of thistemplate resulted in a 252 nt antisense cRNA probe with 66 ntcorresponding to the vector sequence. The BDNF plasmidsequence was checked by sequencing after cloning. This probehybridizes all BDNF transcripts.

Probe labeling for RNAse assay. The RNAse assaywas conducted using 32P-labeled probes complementary toBDNF, Trk B, and 28S rRNA. Trk B mRNA was detectedusing a cRNA complementary to 397 nt (nt 1,327–1,724) of therat Trk B mRNA. This probe, cloned into pGEM3Z byMiddlemas et al. (1991), recognizes full-length and truncatedforms of Trk B mRNA encoding receptors. Plasmids werelinearized with SalI for BDNF and NCoI for Trk B. Eachtemplate (250 ng) was transcribed in the presence of 50 mCi[32aP]UTP (NEN, Paris, France) using T7 polymerase from theSp6/T7 transcription kit (Promega). The 28S probe (115 nt)used as internal control in the RNAse protection assay wastranscribed from pTRI-RNA-28 S (Ambion; CliniSciences,Montrouge, France). Full-length cRNA probes were obtainedusing a Fullengther (TEBU, Yvelines, France) as indicated bythe manufacturer and detailed by Ma et al. (1996). To generatestandard curves, plasmids containing Trk B cDNA and BDNFcDNA were linearized with BstEII (Promega, Madison, WI)and NCoI (Promega), respectively, and sense RNAs were thentranscribed using T3 polymerase (Promega).

Ribonuclease Protection Assay

The RNAse protection assay was performed as previouslydescribed (Ma et al., 1996), with minor modifications. Briefly,total RNA (20 mg) was incubated overnight at 45°C in 30 ml ofhybridization buffer containing 40 mM PIPES, pH 6.4, 400 mMNaCl, 1 mM EDTA, 80% formamide, 500 cpm of 28S antisensecRNA 32P-labeled probe, 1,500 cpm of Trk B antisense32P-labeled probe, and 4.5 3 105 cpm of antisense 32P-labeledBDNF probe. Excess probe, nonspecific endogenous mRNA,and nonspecific hybrids were digested with 350 ml of ribonu-clease solution (10 mM Tris, 300 mM NaCl, 5 mM EDTA, and2 mg/ml of ribonuclease T1) for an additional 60 min at 37°C.RNAse was inhibited by adding 10 ml of 20% sodium dodecylsulfate (SDS) and 3 ml of proteinase K (1 mg/50 ml proteinase Kbuffer: 30 mM Tris, 100 mM NaCl, 1 mM EDTA, and 0.5%SDS, pH 7.4) for 15 min at 37°C. Hybrids were extracted with400 ml Tris-phenol:chloroform/isoamyl alcohol 24:1 and etha-nol precipitated with 70 ml of 7.5 M ammonium acetate and10 mg of tRNA as carrier. Pellets were resuspended in 6 mlRNA loading buffer [80% formamide, EDTA 25 mM, 5 ml each

of 0.4% (w/v) of xylene cyanol and bromophenol blue]. Sampleswere electrophoresed on a 7.1 M urea and 5% polyacrylamidedenaturing gel. Dry gels were exposed to Kodak Bio-Max MRfilms (Eastman Kodak, Rochester, NY) at –80°C with an in-tensifying screen for 3 days. Autoradiographs were analyzed bydensitometry (OD) with a high-resolution Sony CCD XC-77video camera using NIH Image software (W. Rasband; NIH).The ODs of BDNF and Trk B signals were normalized accord-ing to the OD of 28S rRNA. Ratios were expressed as percent-age of control 6SEM. The basal amount of mRNA per samplewas calculated using the corresponding sense RNA standardcurve as reference.

BDNF Immunoassay

The BDNF protein was assayed using the BDNF EmaxImmuno Assay Kit (Promega). This two-site enzyme immuno-assay system (ELISA) has a detection limit of 15 pg/ml, and thecross reactivity with other related neurotrophic factors is ,3%.Protein extractions and assays were conducted according to themanufacturer’s protocol. Briefly, cells were scraped in 150 ml oflysis buffer [137 mM NaCl, 20 mM Trizma base, 1% IGPAL,1 mM phenylmethylsulfonyl fluoride (PMSF), 10 mg/ml apro-tinin, 1mg/ml leupeptin, 0.5 mM sodium vanadate, 10% glyc-erol, and 1% bovine serum albumin (BSA)] and then centrifugedfor 30 min at 10,000g at 4°C. Supernatant (100 ml) was mea-sured with the kit. The intra- and interassay coefficient ofvariations were 3% and 6%, respectively.

RESULTS

Time Course of BDNF mRNA Induction AfterGlutamate Application to Primary Cultures ofHypothalamic Neurons Determined by RNAseProtection Assay

mRNA analysis of hypothalamic neurons showedthat, under basal conditions, cultures contained low levelsof BDNF mRNA (50 6 7 fg/mg of total RNA; n 5 16)compared to Trk B mRNA, which was present in higherquantity (5.8 6 0.4 pg/mg of total RNA; n 5 6). Figure1A shows that the protected fragments were at the ex-pected sizes, corresponding to those protected by ratcRNA probes. Stimulation of hypothalamic cultures with100 mM glutamate at different times induced a rapidincrease in BDNF mRNA expression (Fig. 1A,B). Thiseffect was significant at 30 min (10.5% augmentation vs.control, P , 0.05), increased at 60 and 180 min (25% and41.6% augmentation vs. control, P , 0.0005), decreased at300 min (9.5% augmentation vs. control, P , 0.05), andreturned to basal values at 480 min. With the same sam-ples, we detected a rapid, significant, and transient de-crease in Trk B mRNA content after 15 min of glutamateapplication (Fig. 1A,B; 18% inhibition vs. basal value, P ,0.05).

Dose-Dependent Effect of Glutamate-InducedExpression of BDNF mRNA Determined byRNAse Protection Assay

Hypothalamic neurons were treated for 3 hr with 10,25, 50, or 100 mM glutamate. Three hours of incubation

BDNF Transcripts in Hypothalamic Neurons 379

were chosen because a maximal stimulation of BDNFmRNA expression by glutamate was detected at this time.Figure 2 shows that glutamate induced a dose-dependentincrease in BDNF mRNA expression. This effect wassignificant with 25 mM glutamate (12.9% augmentationvs. control, P , 0.005), and maximal effects were obtainedwith 50 or 100 mM glutamate (32.6 and 41.6% augmen-tation vs. control, P , 0.0005). In contrast, Trk B mRNA

showed a slightly decreased expression, which was non-significant even at the highest glutamate concentrations.

Expression of Different BDNF TranscriptsAnalyzed by RT-PCR

Figure 3A shows a schematic representation of thedifferent BDNF mRNAs transcribed from the BDNF

Fig. 1. Time course of BDNF and Trk B mRNA expression inhypothalamic neurons after glutamate treatment determined by RNAseprotection assay. A: Representative autoradiogram of a gel, illustratingchanges in Trk B and BDNF mRNA content under the differentexperimental conditions. L, ladder; NDP, undigested probes; DP,digested probes. The autoradiogram shows protected RNA fragmentsat the expected sizes at different times (15, 30, 60, 180, 300, and480 min) of 100 mM glutamate application. B: Statistical analysis ofBDNF and Trk B mRNA expressions from different time courses after100 mM glutamate treatment. Ratios of OD of BDNF or Trk BmRNA hybridization signals vs. 28S rRNA hybridization signal in eachsample were calculated. The results are mean 6 SEM of these ratiosexpressed as percentage of control values (considered as 100%). *P ,0.05, **P , 0.005, and ***P , 0.0005 vs. control, nontreated sistercultures (nine or ten determinations from seven different experiments).

Fig. 2. Dose-response curves of BDNF and Trk B mRNA expressionafter glutamate treatment determined by RNAse protection assay.Hypothalamic neurons were incubated for 180 min with differentglutamate concentrations (10, 25, 50, or 100 mM) for 180 min.A: Representative autoradiogram of a gel as indicated in the legend toFigure 1. B: Ratios of OD of BDNF or Trk B mRNA hybridizationsignals vs. 28S rRNA hybridization signal in each sample were calcu-lated. The results are mean 6 SEM of these ratios expressed as per-centage of control values (considered as 100%). **P , 0.005 and***P , 0.0005 vs. control nontreated sister cultures (eight to tendeterminations from four experiments).


gene. We examined the expression of exon I, II, III, andIV BDNF mRNAs in hypothalamic neurons (Fig. 3B)using the RT-PCR method and in adult rat hypothalamustissue as positive control (Fig. 3C). The four types ofBDNF mRNAs were detected; exon III BDNF mRNAwas mainly expressed and exon IV BDNF mRNA wasweakly visible. Figure 3B,C also shows that the amplifiedfragments were of the expected sizes.

Time Course of Glutamate-Induced Expression ofBDNF mRNAs in Hypothalamic NeuronsDetermined by Semiquantitative RT-PCR

To evaluate variations in the expression of the dif-ferent BDNF gene transcripts after glutamate treatment,their expressions were determined relative to that of thecyclophilin mRNA. BDNF transcript expressions wereanalyzed after 15, 30, 60, 180, and 300 min of 100 mMglutamate treatment. Figure 4 shows that all BDNFmRNAs containing different 59 exons were differentiallyincreased after glutamate stimulation. Exon III and IVBDNF mRNAs were rapidly and significantly increased,whereas exon I and II BDNF mRNAs showed moredelayed variations. In fact, after 30 min of stimulation with100 mM glutamate, exon IV BDNF mRNA was increasedby 27.7% (P , 0.005) over the control level, reached apeak after 60 min (43.6% augmentation, P , 0.0005), andbegan decreasing after 180 min of glutamate application(12% augmentation, P , 0.005). At 300 min, valuessignificantly lower than those determined under basalnontreated conditions were obtained (27.5% inhibition vs.control, P , 0.005). Similarly, exon III BDNF mRNApeaked at 60 min of stimulation (45.3% augmentation,P , 0.005) but remained 25.3% (P , 0.0005) and 15.5%(P , 0.05) higher than the control level after 180 and300 min stimulations, respectively. In contrast, increases inexon I and II BDNF mRNAs were slower to develop.Exon I BDNF mRNA increased up to 180 min, reachinga plateau that remained steady up to 300 min (86.3% and85.5% augmentation, respectively, P , 0.0005). Likewise,exon II BDNF mRNA increased slowly, to reach a peakat 180 min (39.8% augmentation, P , 0.0005) and re-mained elevated (17.5%, P , 0.05) at 300 min of treat-ment. These data indicated that exon I BDNF mRNA wasthe most responsive, whereas exon II BDNF mRNA wasthe least responsive in hypothalamic neurons. After480 min of 100 mM glutamate stimulation, exon II, III,and IV BDNF mRNAs returned to basal values, except forexon I BDNF mRNA, which still exhibited strong ex-

Š

Fig. 3. Expression of BDNF mRNAs in hypothalamic neurons deter-mined by RT-PCR. A: Representation of the primers used in PCRanalysis to detect BDNF transcripts in relation to the schematic repre-sentation of the gene. For the gene shown at the top, exons are shownas boxes and introns as lines. The eight possible transcripts from thegene are shown below the scheme, with lines indicating the alternativesplicing sites. Specific primers used for RT-PCR amplification areindicated as solid lines. SI, SII, SIII, and SIV correspond to sense-specific primers for exons I, II, III and IV, respectively, according to thenomenclature proposed by Timmusk et al. (1993). Dotted lines (AS)correspond to the common antisense primer specific to exon V. Thesizes of the amplified fragments for each transcript are indicated be-tween dotted lines. One microgram of total RNA prepared fromprimary cultures of hypothalamic neurons (B) or from adult hypotha-lamic tissue (C) was reverse transcribed as described in Materials andMethods. PCR of the four BDNF transcripts was carried out for 32cycles. The amplified cDNA was visualized by ethidium bromidestaining.


pression (data not shown). Subsequent pharmacologicalexperiments were thus performed at 3 hr for exon I and IIBDNF mRNAs and at 1 hr for exon III and IV BDNFmRNAs.

Dose-Dependent Effect of Glutamate on BDNFmRNAs in Hypothalamic Neurons Determined bySemiquantitative RT-PCR

Neurons were treated with 10, 25, 50, or 100 mMglutamate for 180 min to determine the expression of exonI and II BDNF mRNAs (Fig. 5A). For transcript I, thestimulatory effect of glutamate was already significant at10 mM (14.5% augmentation, P , 0.05), with a markedincrease at 25, 50, or 100 mM glutamate (30.9%, 71.1%,and 86.3% augmentation, respectively, P , 0.0005). Nosignificant differences were observed between 50 and100 mM glutamate applications. In contrast, exon IIBDNF mRNA showed weaker stimulation in comparisonwith exon I BDNF mRNA induction. It was significant at25 mM glutamate (24.6% augmentation, P , 0.05) andhighly significant at 50 or 100 mM (30% and 39.7%augmentation, respectively, P , 0.005). No significantdifferences were observed among 25, 50, and 100 mMglutamate.

Figure 5B shows the stimulatory effect of glutamateon the expression of exon III and IV BDNF mRNAs.These experiments were performed at 60 min, becausethese two transcripts reached maximal stimulation at this

time according to time course studies (see Fig. 4). Nosignificant differences were observed between 50 and100 mM glutamate treatments; augmentations in exon IIIBDNF mRNA were 44.6% and 45.3%, respectively (P ,0.0005), and 34.5% and 43.6%, respectively (P , 0.0005),for exon IV BDNF mRNA. Our overall results indicatedthat in hypothalamic neurons exon I BDNF mRNA wasthe most responsive, whereas exon II BDNF mRNA wasthe least responsive.

Involvement of NMDA or Non-NMDA Receptorsin Glutamate-Induced Expression of BDNFmRNAs Determined by SemiquantitativeRT-PCR

To determine whether NMDA receptors were in-volved in glutamate-induced expression of various BDNFtranscripts, we examined the effects of 1) NMDA onmRNA expression and 2) the noncompetitive antagonistMK-801 on glutamate- or NMDA-induced response.Figure 6A,B shows that NMDA reproduced glutamatestimulation on exon II and IV BDNF mRNAs. Glutamatewas more efficient than NMDA in increasing exon IBDNF mRNA, whereas the contrary was observed forexon III BDNF mRNA. Pretreatment of neurons for5 min with 10 mM MK-801 reversed the stimulationinduced by glutamate or by NMDA (50 mM). MK-801alone slightly but significantly (P , 0.05) inhibited basallevels of exon I and II BDNF mRNAs (17.2% and 23.4%,respectively, vs. control) without affecting exon III and IVBDNF mRNAs. The fact that exon I BDNF mRNA wasmore stimulated by glutamate than by NMDA suggeststhat other glutamate receptor types might be involved inthis effect. Thus, to examine this question, we tested theeffect of 100 mM DNQX (a non-NMDA ionotropicreceptor inhibitor) and of 1 mM MCPG (a metabotropicreceptor inhibitor) on the responses induced by 50 mMglutamate. Figure 6C,D shows that DNQX had no sig-nificant effect on glutamate-induced expression of exon IIand IV BDNF mRNAs, whereas it partially or nonsignifi-cantly inhibited glutamate-induced expression of exon Iand III BDNF mRNAs, respectively. DNQX alone hadno effect on basal expression of these transcripts either.MCPG had no effect on glutamate-induced expression ofany transcript, but, alone, it slightly but significantly (P ,0.05) stimulated basal expression of exon I BDNF mRNAand slightly inhibited basal expression of exon III BDNFmRNA (P , 0.05).

Effect of Cycloheximide and Actinomycin D onGlutamate-Induced Expression of BDNF mRNAsDetermined by Semiquantitative RT-PCR

To determine whether glutamate-induced expres-sion of the different BDNF mRNA transcripts depends onprotein synthesis, we tested the effect of cycloheximide, aprotein synthesis inhibitor. Figure 7A,B shows that pre-treatment with cycloheximide (5 mg/ml) 10 min beforeand during glutamate stimulation significantly inhibitedglutamate-induced expression of exon I and II BDNFmRNAs (P , 0.0005). Cycloheximide alone had no

Fig. 4. Time course effect of glutamate on BDNF mRNAs expressiondetermined by semiquantitative RT-PCR. RT-PCR was carried outas described in detail in Materials and Methods. Each exon was coam-plified, with cyclophilin used as internal standard. Neurons were treatedwith 100 mM glutamate for 15, 30, 60, 180, and 300 min. The resultswere calculated as the intensity of the lane of each transcript over theintensity of the corresponding cyclophilin band and expressed as themean 6 SEM of the percentage of control (considered as 100%). *P ,0.05, **P , 0.005, and ***P , 0.0005 vs. control, nontreated cultures(six to nine determinations from four or five independent experiments).


significant effect on basal expression of these two tran-scripts. In contrast, as shown in Figure 7B, cycloheximideapplication did not suppress glutamate-induced expressionof exon III and IV BDNF mRNAs, whereas, alone, itincreased basal expression of these transcripts by 35.4% and22.8%, respectively (P , 0.005). The stimulatory effects ofcycloheximide and glutamate were additive. Figure 7C,Dshows that treatment with 10 mg/ml actinomycin D, aninhibitor of RNA transcription, 10 min before glutamateapplication fully and significantly (P , 0.005) inhibitedglutamate-induced expression of all BDNF mRNA tran-scripts. Similarly, treatment with actinomycin D aloneinhibited basal expression of exon I, II, III, and IV BDNFmRNAs by 17.6% (P , 0.0005), 18.1% (P , 0.0005),19.8% (P , 0.005), and 16.9% (P , 0.05), respectively.

Time Course of Glutamate- and NMDA-InducedExpression of BDNF Protein Content Determinedby ELISA

To determine whether the increased expression ofBDNF mRNA by glutamate and NMDA results in an

increase in protein levels, we performed a time coursestudy with 100 mM glutamate or 50 mM NMDA. Figure8A,B shows BDNF cellular content after 180, 360, and480 min of glutamate and NMDA treatments, respec-tively. Basal BDNF content was 64.1 6 3.5 pg/dish (n 59). Figure 8A shows that maximal BDNF augmentationwas obtained after stimulating cells for 180 min with100 mM glutamate: 99 6 2.95 pg/dish (n 5 4, P ,0.001). At 360 and 480 min, BDNF content slightlydecreased (88 6 2 pg/dish, n 5 4, P , 0.01 and 86 62 pg/dish, n 5 4, P , 0.001, respectively). NMDAtreatment increased BDNF levels to 103.2 6 5.8 pg/dish(n 5 5, P , 0.001) after 180 min stimulation. After 360 minof stimulation, BDNF content continued to increase,reaching a plateau at 480 min; the results were 142.8 6 8.1and 145.8 6 6.3 pg/dish (n 5 5, P , 0.001), respectively.

DISCUSSIONIn the present study, using exon-specific probes, we

demonstrate that glutamate exerts a differential time courseregulation of multiple BDNF transcripts, with a rapid and

Fig. 5. Dose-response curves of glutamate-induced BDNF mRNAs determined by semiquantitativeRT-PCR. Effect of glutamate on the expression of exon I and II BDNF mRNAs (A) and exon III andIV BDNF mRNAs (B). Neurons were incubated with the different glutamate concentrations for 3 hr forexon I and II BDNF mRNAs and for 1 hr for exon III and IV BDNF mRNAs. The results are expressedas indicated in the legend to Figure 4. *P , 0.05, **P , 0.005, and ***P , 0.0005 vs. control nontreatedcultures (eight or nine determinations from four or five independent experiments).


transitory decrease of Trk B mRNA expression in rathypothalamic neurons. In the present paradigm with fetalhypothalamic neurons, BDNF transcripts from exons I, II,III, and IV were expressed. Exon III BDNF mRNA seems

to be the most abundant in that it was reverse transcribedfrom one-tenth of total mRNA compared with the othermRNAs. We found a similar pattern of BDNF mRNAexpression in adult hypothalamic tissue (Marmigere et al.,

Fig. 6. Effects of glutamate, NMDA, MK-801, and non-NMDA glu-tamate receptor antagonists on BDNF mRNAs determined by semi-quantitative RT-PCR. A: Effect of glutamate (50 mM), NMDA(50 mM), MK-801 (10 mM), and MK-801 1 the agonists on exon I andII BDNF mRNAs. Neurons were incubated for 3 hr with the differentsubstances. B: Same experiments for exon III and IV BDNF mRNAs,but neurons were incubated with the different substances for 1 hr.C: Effects of 1 mM DNQX or 100 mM MCPG alone or on 50 mM

glutamate-induced expression of exon I and II BDNF mRNAs. Neu-rons were incubated with the different substances for 3 hr. D: Sameexperiments for exon III and IV BDNF mRNAs, but neurons weretreated for 1 hr. The results are expressed as indicated in the legend toFigure 4. *P , 0.05, **P , 0.005, and ***P , 0.0005 vs. controlnontreated cultures. 11P , 0.005 and 111P , 0.0005 vs. glutamate-treated cultures (four to eight determinations from three to five differ-ent experiments).


1998), indicating that, in hypothalamic neurons, the ex-pression patterns of transcripts described here were due notto the early developmental stage analyzed but to a tissue-related specificity. Our data supply additional evidencedemonstrating a differential promoter usage for BDNFgene expression in brain. These data are consistent withthose reported for other CNS regions, e.g., mainly detec-

tion of BDNF mRNAs containing exons I, II, and III inthe hippocampus and cerebral cortex (Timmusk et al.,1993; Metsis et al., 1993; Bishop et al., 1994). Alternativepromoters are commonly used to express the same geneproduct at different stages of development or in differentcell types (Schibler and Sierra, 1987; Shapiro et al., 1991).Other examples of alternative promoter usage are illus-

Fig. 7. Effect of cycloheximide or actinomycin D treatments on glu-tamate induction of BDNF mRNAs in hypothalamic neurons. Cyclo-heximide (5 mg/ml) or actinomycin D (10 mg/ml) were added 10 minbefore and during 180 min of glutamate (50 mM) stimulation for exonI and II BDNF mRNAs (A,C) and 10 min before and during 1 hr ofglutamate stimulation for exon III and IV BDNF mRNAs (B,D). Note

that actinomycin D fully inhibited glutamate-induced expression of allBDNF mRNA transcripts. The results are expressed as indicated in thelegend to Figure 4. *P , 0.05, **P , 0.005, and ***P , 0.0005 vs.control, nontreated cultures. 11P , 0.005 and 111P , 0.0005 vs.glutamate-treated cultures (six or seven determinations from threeindependent experiments).


trated by the gene of the human gonadotropin-releasinghormone, for which one promoter is active in neuronaltissue and a second is active in reproductive tissue (Donget al., 1997), or that of the gene for rat growth hormone-releasing hormone, with one promoter active in the hy-pothalamus and the second one active in the placenta(Gonzales-Crespo and Boronat, 1991). Different promoterusage in prolactin receptor gene expression has also beendescribed for rat liver, ovary, and mammary gland (Mold-rup et al., 1996).

Interestingly, after glutamate treatment, the expres-sion of all four transcripts was increased in hypothalamicneurons with different time courses. In these neurons,total BDNF mRNA might represent a combination of thefour different transcripts, as reported for the hippocampus.In addition, BDNF exon I was the most inducible brainpromoter, with a time course induction similar to thatdescribed for hippocampus of adult animals after kainicacid injections (Timmusk et al., 1993; Metsis et al., 1993).

Comparison of the time course of increases in exon-specific mRNAs revealed that maximal effects for exon Iand II BDNF mRNAs were observed after 3 hr of gluta-mate application and after 1 hr for exon III and IV BDNFmRNAs. This feature suggests that promoters I and II arelocated close to each other within the BDNF gene and,therefore, may share regulatory sequences (Timmusk etal., 1993). These data also suggest different sequential rolesfor these exons in the translation process, to adjust BDNFlevels to respond to specific stimuli. Exon I and II BDNFmRNAs remained elevated for longer times after gluta-mate treatment than exon III and IV BDNF mRNAs. Thesame kinetic profile for these promoters has been found invivo for adult hippocampus after kainic acid injections(Metsis et al., 1993) or for BDNF exons III and IVfollowing electric stimulation (Lauterborn et al., 1996).Our results also show that, unlike the case with exon I andII BDNF mRNAs, promoters linked to exon III and IVBDNF mRNAs are fully inducible in the presence of theprotein synthesis inhibitor cycloheximide, suggesting thatthese transcripts are regulated as early genes. In addition,cycloheximide alone increased exon III and exon IVBDNF mRNA expression. In agreement with these data,previous studies have demonstrated that protein synthesisinhibitors superinduce immediate early gene mRNAs(Edwards and Mahadevan, 1992). It has been already re-ported that BDNF is induced as an immediate early genefollowing NMDA receptor activation in the hippocampus(Hughes et al., 1993), an effect antagonized by MK-801.In our model, the effect of cycloheximide was additivewith respect to that induced by glutamate, suggesting thatthe two effects were independently regulated. Resultsobtained in the presence of actinomycin D led us toconclude that the effect of glutamate on BDNF transcriptexpression was due to a transcriptional effect on the gene.Taken together, these data support a physiological regu-latory role of glutamate in hypothalamic neurons, insofaras increase of BDNF mRNA results in protein augmen-tation.

The glutamate stimulatory effects seemed to be es-sentially mediated by NMDA receptor activation in thatthe mRNA increases were completely blocked by MK-801. These data are consistent with findings showing thatactivation of NMDA receptors in vivo increases BDNFmRNA expression in hippocampal formation (Gwag andSpringer, 1993) or in cultured cerebellar granule neurons(Favaron et al., 1993). However, in these reports, onlytotal BDNF mRNA expression was studied, without an-alyzing mRNA transcribed from different promoters. In-terestingly, MK-801 alone significantly inhibited basal lev-els of mRNA containing exons I and II. Thus, resultssuggest that glutamate is tonically released in the incuba-tion media, thereby settling basal transcription of thesetranscripts through NMDA receptor activation. In con-trast, a similar effect is observed for mRNA containingexon III, but after MCPG addition, suggesting the in-volvement of metabotropic receptors in maintaining basallevels of this transcript. These different regulations could

Fig. 8. Time course effect of glutamate or NMDA on BDNF proteincontent determined by ELISA. Cultures were treated with 100 mMglutamate (A) or 50 mM NMDA (B) for 180, 360, or 480 min. Theresults are calculated in picograms per dish according to the internalstandard curve and expressed as mean 6 SEM. *P , 0.01 and **P ,0.001 (four or five determinations).


be explained either by a differential expression of gluta-mate receptors in different cellular populations presentingonly some promoters or by different glutamate receptorsinvolved in the regulation of BDNF promoters. Indeed,previous studies have demonstrated the critical role playedby glutamate through NMDA (Tapia-Arancibia et Astier,1988; Benyassi et al., 1991; Gu et al., 1999) and non-NMDA ionotropic (van den Pol et al., 1990) or metabo-tropic receptors in the hypothalamus, both in the devel-oping and in the adult brain (van den Pol et al., 1994,1995; Ghosh et al., 1997). Moreover, a developmentallyregulated expression of NMDA (Rage et al., 1994) ormetabotropic glutamate receptors in the hypthalamus hasbeen reported (Ghosh et al., 1997). However, the kineticprofiles obtained with glutamate and NMDA on proteincontent were a bit different. The longer lasting markedeffect of NMDA in comparison with that induced byglutamate could be attributed to a longer action ofNMDA, because no natural reuptake or degradation sys-tems exist for this molecule. Another explanation is thatglutamate subsequently activates inhibitory mechanisms ofthe transcription that occurs via non-NMDA pathways.

Previous studies also conducted on hypothalamicneurons allowed us to demonstrate that the stimulation ofionotropic NMDA receptors increases intracellular Ca21

concentrations in a dose-dependent manner (Dayanithi etal., 1995). In addition, it is known that the influx ofextracellular Ca21regulates BDNF mRNA levels (Shieh etal., 1998; Tao et al., 1998; Takeuchi et al., 2000). Theseauthors reported evidence that upstream regions of exonIII and IV contain calcium-responsive elements able tobind transcription factors activated by Ca21 influx such ascAMP response element-binding protein (CREB) orclosely related family members. These observations pro-vide a link between electrical activity, Ca21 influx, andBDNF mRNA up-regulation. In the present work, Ca21

entry into neurons via the NMDA subtype of glutamatereceptors might induce CREB phosphorylation andBDNF transcription.

Finally, after treating neurons with glutamate, Trk BmRNA expression was rapidly (15 min) and transientlydecreased and returned to control values 30 min later.Because our probe recognizes all Trk B isoforms, we canalso assume that the catalytic form is the predominantisoform in these developing neurons, as has been alreadyreported (Escandon et al., 1994; Allendoerfer et al., 1994).In cortical (Knusel et al., 1997) or hippocampal (Frank etal., 1996) neurons in culture, Trk B mRNA has beenshown to be down-regulated by increased concentrationsof its ligand, BDNF. This has been explained in terms offunctional desensitization to BDNF. Although in thepresent work the mRNA encoding BDNF measured inparallel in the same experiments was not significantlyincreased at 15 min, ready intracellular pools of BDNFprotein might be secreted after glutamate stimulation, thusinducing receptor desensitization. In support of this hy-pothesis, it has been demonstrated that glutamate substan-

tially and very rapidly induced BDNF release from hip-pocampal slices or cultured neurons (Canossa et al., 1997).

We have already demonstrated the presence ofmRNA encoding Trk B receptors in cultured hypotha-lamic neurons (Rage et al., 1999) as well as the function-ality of these receptors. Their activation by its cognateligand (BDNF) induces short-lasting (Marmigere et al.,2001) and long-lasting (Rage et al., 1999; Loudes et al.,1999, 2000) effects. Thus, BDNF was able to induce rapidstimulatory effects on somatostatin release (Marmigere etal., 2001) and also increased somatostatin synthesis (Rageet al., 1999; Loudes et al., 1999, 2000). Because glutamateinduces similar effects on hypothalamic neurons (Tapia-Arancibia and Astier, 1988; Rage et al., 1993), it could behypothesized that BDNF mediates these short- and long-lasting effects on somatostatin neurons, insofar as timecourses are compatible with such actions. A similar cor-relation between somatostatin and BDNF expressions hasbeen reported for the hippocampus following kindlingstimulation (Ernfors et al., 1991; Pretel et al., 1995) orduring aging in the primate brain (Hayashi et al., 1997),providing an experimental substrate to our hypothesis.

In conclusion, this study is a first step toward under-standing the regulatory mechanisms governing BDNFgene expression at the early stages of neuronal develop-ment in hypothalamic neurons. These data suggest thatglutamate, through NMDA receptor activation andBDNF up-regulation, may indirectly exert trophic activityin developing hypothalamic neurons. Given that in theadult rat both BDNF and glutamate are associated with thehypothalamic stress response, the findings reported heremight provide some clues to an understanding of plasticityphenomena occurring beyond fetal life.

ACKNOWLEDGMENTSF.M. was supported by a grant from the French

“Ministere de l’Education Nationale, de la Recherche etde la Technologie.” F.R. was supported by a grant fromthe CNRS (Soutien aux Jeunes Equipes). We thank Dr.Sandor Arancibia for critical reading of the manuscript andMr. Edmond Savary for technical assistance.

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