Involvement of VIP on BDNF-induced somatostatin gene expression in cultured fetal rat cerebral cortical cells

Molecular Brain Research 94 (2001) 59–66 www.elsevier.com / locate / bres

Research report

Involvement of VIP on BDNF-induced somatostatin gene expression in cultured fetal rat cerebral cortical cells b a ´ ´ Gemma Villuendas a , Franco Sanchez-Franco , Nuria Palacios a , Miriam Fernandez , a, Lucinda Cacicedo * a

´ , Hospital Ramon ´ y Cajal, Carretera de Colmenar, Km 9, 28034 Madrid, Spain Servicio de Endocrinologıa b Hospital Carlos III, 28029 Madrid, Spain Accepted 12 June 2001

Abstract Previous studies have shown that BDNF promotes expression of SS. In earlier studies we demonstrated the stimulatory effect of locally produced VIP upon SS secretion. These facts led us to explore the peptidergic action of BDNF on VIP, and to determine if BDNF-induced SS might be mediated by the induction of VIP. Cultured fetal rat cerebralcortical cells were incubated with BDNF (50 ng / ml) and / or VIP (10 211 M) for 2 and 5 days. In other experiments IgGs from BDNF or VIP antisera were also added. BDNF increased VIP and SS gene expression and peptide production. After 2 days of incubation with both BDNF and VIP the induction of SS mRNA was similar to that obtained with BDNF alone. However when the treatment was extended to 5 days the increase in SS mRNA was higher than that obtained with BDNF alone. This finding suggests the possibility that both factors acted synergistically. To define the potential role of VIP in the response of SS gene expression to BDNF, endogenous VIP was blocked with IgGs from VIP antiserum. Under these experimental conditions BDNF-induced SS decreased. Our study provides the first evidence that BDNF up-regulates VIP gene expression and concentration of the peptide. The involvement of VIP on BDNF-induced SS gene expression is also demonstrated.  2001 Elsevier Science B.V. All rights reserved. Theme: Development and regeneration Topic: Neurotrophic factors: biological effects Keywords: Somatostatin; BDNF; VIP; Neuronal activity; Neuropeptide; Neurotrophin

1. Introduction During development, each individual neuron is related to a determined set of neurotransmitters and neuropeptides, which are released into synapses to pass correct signals to target cells. Production of neuropeptides is dynamically influenced by diffusible protein factors, including cytokines that are synthesized by many cell types [38]. Previous studies have demonstrated that production of neuropeptides in peripheral neurons [11,24,35,42] can be influenced by neurotrophic factors. More recently, it has been found that neurotrophins also have strong peptidergic

*Corresponding author. Tel.: 134-91-336-8547; fax: 134-91-3369016. E-mail address: [email protected] (L. Cacicedo).

activities in the central nervous system (CNS) both in vivo [37] and in vitro [5,26,36,41]. Brain-derived neurotrophic factor (BDNF) and its receptor trkB are predominantly expressed in the CNS [15,21,23,48] in particular in the hippocampus and cerebral cortex, [28]. Its functions as a promoter of neuronal survival [7,2,10,18,44,] and differentiation [43,47] suggest that BDNF might play an important role in the developing brain. This neurotrophic factor has a potent neuronal differentiation activity in vivo [5,37] and in vitro [5,26,36,41] that increases levels of several neuropeptides such as somatostatin (SS). Many studies have provided experimental evidence that neuropeptide expression in the CNS can be affected by neuronal activity [1,17,25,31,45,46]. Neurotrophic factors are known to mediate such alterations in neuropeptide expression in the peripheral nervous system (PNS) [14,42]. The influence in the CNS of neurotrophins on the regula-

0169-328X / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved. PII: S0169-328X( 01 )00177-2

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tion of NPY [30] and SS [26] by neuronal activity has also been proved. The neuropeptide vasoactive intestinal (VIP) is expressed in the CNS during development [39] where it has been shown to exert neurotrophic actions such as an increase of neuronal survival [4] and promotion of embryonic growth [16]. Moreover, VIP stimulates the expression of BDNF in cortical astrocytes and neuronal cells [39]. We have demonstrated that endogenous VIP upregulates SS secretion by cultured fetal rat cerebral cortical and hypothalamic cells [9]. In view of these facts and the existence of a functional interaction between neuropeptides in the CNS, our aim in this work has been to determine if the action of BDNF on SS might be mediated by the induction of VIP gene during nervous system development. Here we report a novel interplay between BDNF and VIP in the neuronal differentiation activity on cultured cerebral cortical neurons.

the third day in vitro (DIV), and experiments were performed 2–3 days afterwards.

2.3. BDNF incubation After 5–6 DIV, media were removed and fetal rat cerebral cortical cells were incubated in defined medium in the presence or not of BDNF (20 and 50 ng / ml) for 2 and 5 days. At the end of incubation, cells and media were processed and used for immunoreactive somatostatin (IRSS) or immunoreactive VIP (IR-VIP), and SS or VIP mRNAs determination.

2.4. VIP incubation

2. Material and methods

After 5–6 DIV media were removed and fetal rat cerebral cortical cells were incubated in defined medium in the presence or not of VIP (10 27 and 10 211 M). VIP was added every 24 h for five consecutive days in the experiments shown in Fig. 5. At the end of incubation, cells were processed and used for SS mRNA determination.

2.1. Buffers and media

2.5. VIP blocking studies

Hank’s balanced salt solution (HBSS) and minimum essential medium (HMEM) were obtained from Biochrom (Berlin, Germany). HMEM supplemented (HMEMs) consisted of HMEM containing 10% fetal calf serum (FCS), 10% horse serum (HS), streptomycin and penicillin (100U / ml) and L-glutamine (200 mg / ml) from BioWhittaker (Walkersville, MD, USA), glucose (6 g / l) and insulin (80 mU / ml) from Sigma (St. Louis, MO, USA). Defined medium consisted of DMEM (glucose 1 g / l): (1:1) Ham’s F12 supplemented with: NaHCO 3 (1.2 g / l) and glucose (6 g / l) from Merck (Darmstadt, Germany), transferrin (0.1 mg / ml) (Boehringer Mannheim, Mannheim, Germany), putrescine (10 25 M) and sodium selenite (2310 28 M), corticosterone (10 27 M), Hepes (15 mM), T3 (10 210 M) and insulin (80 mU / ml) from Sigma, L-glutamine (4 mM) and penicillin–streptomycin (100 U / ml) from BioWhittaker and bFGF (25 ng / ml) (Genzyme Diagnostics, Cambridge, MA, USA).

After 5–6 DIV, media were removed and replaced by defined medium containing BDNF (50 ng / ml), VIP (10 211 M) or both. IgGs from specific polyclonal antibodies to BDNF (BDNF-Ab) (35 mg / 1.5 ml) or to VIP (VIP-Ab) (35 mg / 1.5 ml) obtained in our laboratory were added to the media in the presence of BDNF (50 ng / ml), and the cells were incubated for time periods of 2 and 5 days. All the substances tested were added only once, at the beginning of the experiments. Controls received media alone. We had previously tested that IgGs from normal rabbit serum did not alter SS mRNA levels.

2.2. Cell culture Preparation of primary long-term dispersed cell cultures of fetal rat cerebral cortex was done as previously described, [8]. Timed pregnant Wistar rats were raised in our laboratory. On embryonic day 17, the embryos were removed from the ether-anesthetized mothers. The cerebral cortices were dissected under sterile conditions and the cells mechanically dispersed. The cells were suspended in HMEMs, plated in culture dishes at a density of 5–6310 6 cells / 35 mm plate, and kept in a humidified atmosphere of 5% CO 2 , 95% air at 378C. The medium was changed on

2.6. Preparation and purification of antibodies BDNF-Ab was raised in our laboratory. rhBDNF (1 mg), donated by Amgen (Thousand Oaks, CA, USA), was emulsified with 1.5 ml complete Freund’s adjuvant and 2 mg dried tubercle bacilli and injected intradermally in adult male rabbits. Each animal also received 0.5 ml pertussis vaccine (s.c.) with the primary immunization. Booster injections were carried out at 2-week intervals. The specificity of the antibody against BDNF was checked by an immunoblotting procedure. BDNF-Ab immunoreactive proteins were visualized by a chemiluminescence detection system (Amersham, Buckinghamshire, UK). BDNF-Ab crossreacted with recombinant human BDNF but not with mouse NGF, human neurotrophin NT-3 or human neurotrophin NT-4 (Alomone Labs, Jerusalem, Israel) (Fig. 1). VIP-Ab against porcine VIP has been characterized in previous studies [27]. IgGs were purified from rabbit antisera against recombinant human BDNF

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Fig. 1. Analysis of the specificity of BDNF-Ab. For Western blotting 50 and 5 ng of each neurotrophin were loaded in the respective lanes. Lanes: (1,2) rhBDNF; (2,3) mNGF; (4,5) hNT-3; (6,7) h NT-4.

(rhBDNF) or VIP using DEAE-cellulose columns as previously described [9].

2.7. mRNA analysis Total RNA was isolated by the method described by Chomczynski and Sacchi [6]. Northern analysis of SS and VIP mRNAs was performed as follows. Total RNAs were electrophoresed in 1% agarose–formaldehyde gels, followed by electrotransfer to nylon membranes (Nytran, Schleicher and Schuell, Keene, NH, USA) and UV crosslinking (Hoefer Scientific Instruments, San Francisco, CA, USA). SS complementary DNA (cDNA) in the pSP65 vector [38] was used to produce a 32 P-labeled antisense RNA probe after SalI linearization and SP6 RNA polymerase synthesis, according to the Riboprobe Kit method (Promega, Madison, WI, USA) as previously described [45]. The VIP cDNA was subcloned into the expression vector pSP64 and linearized using BamHI [13]. A 32 Plabeled antisense RNA probe was synthesized with SP6 polymerase. Hybridizations were carried out overnight at 658C as previously described [45,22], after adding 6310 6 cpm / ml of labeled SS and VIP probes, for message measurement. After hybridization, the blots were washed twice for 30 min in 23 SSC, 0.1% SDS at 658C and twice for 30 min in 0.23 SSC, 0.1% SDS. Autoradiograms were developed after exposure to X-ray film (Kodak X-Omat, Eastman Kodak, Rochester, NY, USA) at 2808C using two intensifying screens, for different periods of time according to the type of probe used. Equal loading was confirmed by comparing intensities of ethidium bromide-stained ribosomal 28S RNA in the nylon filter. The intensities of bromide-stained filters and autoradiogram signal levels were quantified by densitometric scanning using AdobePhotoshop 2.0 (Adobe Systems, Mountain View, CA, USA) and NIH-Image 1.47. Data were expressed as arbitrary units after correction for the 28S ribosomal band.

tiserum was 1:300 000, and the cross-reaction with SS-28 was 20%. The assay sensitivity was 1 pg / tube. The intra– interassay variations were 8 and 15%, respectively. Immunoreactive VIP (IR-VIP) was quantified by a well characterized RIA [27], using an antiserum against pVIP raised in our laboratory. The initial dilution of the antibody was 1:200 000. The doubled antibody method for separating bound from free fraction was used.

2.9. Statistical analysis Data are expressed as mean6S.E.M. of three independent experiments from three separate batches of cultures. Tests for significance between sample groups were performed with a two-tailed t-test. For multiple comparisons, ANOVA was used with the Fischer test for post hoc comparisons. Significant differences in expression levels presented as percent controls are based on significant differences in absolute values of message expression. Differences were considered significant if P,0.05.

3. Results

3.1. Effect of BDNF on VIP and SS mRNA levels To investigate the possibility that BDNF might stimulate SS and VIP genes expression on similar cell populations in similar experimental conditions, fetal cerebral cortical cells were cultured with BDNF (20 and 50 ng / ml) for 2 and 5 days. Northern blot hybridization assays were performed to analyze VIP and SS mRNA expression. As shown in Fig. 2, BDNF elicited the induction of VIP and SS mRNA levels. This effect was dose related and more evident after 5 days of treatment.

3.2. Effect of BDNF on media and intracellular VIP and SS content

2.8. SS and VIP radioimmunoassays Immunoreactive SS (IR-SS) was quantified by a wellcharacterized RIA using an antiserum against SS-14 as previously described [8]. The final dilution of the an-

Because SS and VIP mRNA levels were increased upon exposure of cortical neurons to BDNF, we examined whether BDNF stimulation might also lead to an increase in peptide content. Cell exposure to BDNF (50 ng / ml) for

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Fig. 3. Effect of BDNF on media and cell extract SS and VIP content. Cells were incubated in defined medium in the presence or not of BDNF (50 ng / ml) for 2 (SS and VIP) and 5 days (VIP).The values represent the mean6S.E.M. (n53), *P,0.05, **P,0.01. Fig. 2. Induction of VIP and SS mRNA by BDNF. Cells were exposed to BDNF (20 and 50 ng / ml) in defined medium for 2 and 5 days. Densitometric analysis of VIP and SS mRNA values normalized to 28S-ribosomal. Data are expressed as percentage of the control group. Values represent the mean6S.E.M. (n53), *P,0. 05, **P,0.01, ***P, 0.001.

2 days resulted in a significant stimulation of media and intracellular IR-SS content (Fig. 3). The time course of the level of media IR-VIP content showed a higher increment after 5 days in culture with BDNF (50 ng / ml). The intracellular IR-VIP content was not significantly modified, indicating that the increase in media VIP did not cause a depletion in the content of the peptide (Fig. 3). In this context, the results suggest that BDNF enhances VIP production, thus confirming the data shown above (Fig. 2), where BDNF increased VIP mRNA to a maximum level after 5 days in culture, which is comparable with the induction observed at peptide content.

3.3. Involvement of VIP on BDNF-induced SS mRNA expression The results shown above indicate that gene expression and peptide levels of VIP and SS are positively regulated by BDNF in cultured fetal rat cerebral cortical cells (Figs. 2 and 3). Additionally, previous reports from our laboratory [9] demonstrated that locally secreted VIP has a

stimulatory action on SS secretion at this time of brain development. These findings prompted us to investigate whether cortical SS gene expression might be, in turn, regulated by a complex interplay between both factors, BDNF and VIP. To determine the possible physiological role of endogenous VIP in the induction of SS gene expression by BDNF, cortical cells were exposed for 2 and 5 days to various experimental procedures. Some cells were treated with BDNF (50 ng / ml), VIP (10 211 M) or both simultaneously. At 2 days of incubation, both BDNF and VIP increased SS mRNA levels (Fig. 4). The simultaneous presence of both factors did not promote an SS gene induction above that obtained with BDNF alone, suggesting the absence of a synergistic interaction between BDNF and VIP in the regulation of SS. In contrast, the treatment with both factors for a longer period (5 days) elicited a substantial increase in SS mRNA expression, higher than that obtained with each factor separately. This finding led us to investigate the potential role of VIP in the response of SS gene expression to BDNF induction. This was assessed by blocking the effects of VIP with specific VIP-Ab in those cells kept in culture with BDNF for 2 and 5 days (Fig. 4). After 2 days in culture, the levels of SS gene expression partially decreased in the presence of VIP-Ab, compared to those obtained in cultures treated with BDNF alone. This effect

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Fig. 4. Involvement of VIP on BDNF-induced SS gene expression. Cells were exposed for 2 and 5 days to various experimental procedures. Some cells were treated with BDNF (50 ng / ml), VIP 10 211 M or both simultaneously. Other cells were treated with BDNF in the presence of VIP-Ab (35 mg IgG / 1.5 ml) or BDNF-Ab (35 mg IgG / 1.5 ml). Densitometric analysis of SS mRNA values normalized to 28S ribosomal. Data are expressed as percentage of the control group. Values represent the mean6S.E.M. (n53), *P,0.05, **P,0.01, ***P,0.001.

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Fig. 5. Induction of SS mRNA by VIP. Cells were exposed to VIP (10 27 211 and 10 M) in defined medium for 5 days. VIP was added daily. Densitometric analysis of SS mRNA values normalized to 28S ribosomal. Data are expressed as percentage of the control group. Values represent the mean6S.E.M. (n53), *P,0.0 5, ***P,0.001.

4. Discussion was more evident after 5 days in culture, where the presence of VIP-Ab markedly prevented SS gene induction by BDNF. The addition of BDNF-Ab completely blocked BDNF-induced SS mRNA. In previous experiments we confirmed that neither VIP nor BDNF antibodies altered basal SS mRNA expression. Taken together, these results indicate that VIP might mediate the response of SS gene expression to BDNF induction.

3.4. Effect of VIP on SS mRNA levels The fact that the levels of IR-VIP in the media, measured 5 days after its addition to the cultures, were quite similar to those in untreated cultures (Table 1) might explain the lack of effect of VIP on SS mRNA levels that was observed after 5 days of VIP addition. To confirm this hypothesis, cells were treated with a daily dose of VIP for five consecutive days, instead of one dose at the beginning of the experiment. Under these experimental conditions, VIP increased SS mRNA levels and the effect was more evident with the lowest dose of 10 211 M (Fig. 5). Table 1 IR-VIP content in the culture media Treatment

IR-VIP (pg / mg protein)

None VIP BDNF VIP1BDNF

23406315 28506240 (ns) 68406341 a 82806613 a

After 5–6 (DIV) cultures were treated for 5 days with VIP (10–11 M) and BDNF (50 ng / ml). The test substances were added only once, at the beginning of the experiment. Data express the mean6S.D. of three experiments. a P,0.01; ns, not significant versus control.

In this paper we present evidence that BDNF upregulates the expression of VIP mRNA and the content of the peptide and we also confirm the neuropeptidergic action of BDNF on somatostatin gene expression in fetal cultured cerebral cortical cells. The upregulation of SS mRNA induced by BDNF is partially inhibited by VIP antibody, thus the final induction of somatostatin by BDNF might be the result of its induction by BDNF along with the induction of VIP. Several physiological studies have demonstrated that neuropeptide expression in the CNS can be affected by substances of different natures such as drugs and hormones [20] and by neuronal activity [25,45]. The effects of drugs, hormones or electrical activity observed in the CNS might also be in part mediated by these neurotrophic factors. TrkB ligands synthesized by pyramidal neurons influences neuropeptide protein expression in a paracrine or transsynaptic manner [15]. Blockade of endogenous tyrosine kinase neurotrophin receptors prevents the effects of bicuculline on neuropeptide Y (NPY) and somatostatinimmunoreactive neurons [30]. Also it has been hypothesized that hippocampal interneurons might regulate their own neuropeptide pattern through the regulation of BDNF synthesis in, and release from, their target neurons [29].

4.1. Effect of BDNF on VIP gene expression and peptide content Increasing evidence supports a role for VIP in trophic processes during CNS development and neuronal growth [4,16,34]. The neuropeptide VIP stimulates proliferation in human neuroblastoma cells, differentiation and neuronal survival [40], thus conferring an important trophic and developmental function early in ontogeny. The growth

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regulatory actions of VIP are most likely significant when trophic factors are present in extracellular media [34]. VIP stimulates BDNF mRNA in primary cultures of cortical neurons and astrocytes by potentiating the effect of glutamate [39]. Studies from our group demonstrated that VIP gene expression and secretion were regulated by insulinlike growth factor I (IGF-I) [22]. These findings together with the fact that BDNF has a potent peptidergic activity in the brain [36], prompted us to consider if certain trophic actions of BDNF during nervous system development might be mediated through the induction of VIP. Data shown in this study demonstrate that BDNF stimulated VIP gene expression and concentration of the peptide in a dose- and time-dependent manner. These effects were higher 5 days after the onset of the stimulation. Several reasons might account for this late stimulation. Differential expression of TrkB, related to the maturation stage, could change responsiveness to BDNF. Age-dependent response of SS [36] or NPY [30] to BDNF has been shown. BDNF is able to up-regulate the expression of TrkB receptor [12]. Therefore, it is reasonable to speculate that an increased expression of TrkB receptor after prolonged exposure to BDNF might contribute to potentiate its effect on VIP. Moreover, as exogenous VIP has been shown to upregulate BDNF expression by potentiating the effect of glutamate [39] a possible positive feedback between BDNF–VIP–BDNF might account for the high levels of VIP mRNA and VIP content that were observed 5 days after beginning stimulation with BDNF. The marked increase in media VIP content that was observed after 5 days of exposure to BDNF was not accompanied by a depletion in the intracellular content. This finding indicates that an active synthesis of the peptide had taken place under BDNF treatment which is in agreement with the temporal pattern of VIP mRNA response to BDNF stimulus that we found. This study implicates another neuropeptide in the group of neuropeptides whose induction is promoted by neurotrophins, and confirms the hypothesis that interneurons in general require continuous phenotypic support from pyramidal cells [29]. These findings suggest that certain trophic functions of BDNF in different developmental stages of the central nervous system might be the result of the induction of VIP gene.

4.2. Effect of BDNF on SS gene expression and peptide content BDNF promotes expression of various neuropeptides. In cultured cerebral cortical neurons, BDNF increases SS mRNA levels to an extent comparable with its induction observed at peptide levels [35] and enhances differentiation of somatostatin neurons in hypothalamic cultures [26]. Furthermore, several in vivo studies have proved the effect of BDNF on SS gene up-regulation [37]. This study confirms the involvement of BDNF in the regulation of somatostatinergic neurons as previously

reported [5,26,36,37,41]. The increase in SS secretion observed in this study was smaller than that reported by Nawa et al. [36]. The explanation for this difference could be related with the stage of development of the embryos or the culture conditions. We used cerebral cortices of embryonic day 17 rats instead of day 19 rats. In our cultures, glial and neuronal cells were present, whereas their work was done in glial-free cultures. An important point is that unlike Nawa et al. we expressed the amount of media SS as pg / mg of protein instead of total media peptide content. We have observed that in our cultures BDNF and VIP increased the proliferation of cells as measured by PCNA and [ 3 H]thymidine incorporation (manuscript in preparation). Consequently, the media SS peptide content appears to be smaller when data are expressed considering the increment in cell number that occurs. Our results are in correlation with the high abundance of TrkB receptors in somatostatin interneurons from the cerebral cortex [15].

4.3. Involvement of VIP on BDNF-induced SS gene expression Members of the neurotrophin family, utilizing distinct receptor systems and signaling mechanisms, can interact synergistically with other neurotrophic factors or neuropeptides in modulating specific response during embryonic development [19,15]. In this paper we present evidence that confirms the activity of BDNF on VIP. In earlier studies we demonstrated the stimulatory effect of VIP upon SS secretion in fetal cerebral cortical cells in culture [9]. The lack of effect of VIP on SS mRNA induction that was observed 5 days after the addition of VIP could be due to the degradation of exogenous VIP as the levels of IR-VIP in the VIP-treated cultures were quite similar to those found in the control (Table 1). This argument is reinforced by the fact that VIP, when added daily, was able to upregulate SS mRNA levels (Fig. 5). In earlier studies we found that, in cultures with bacitracin, [ 125 I]VIP was degraded up to 90% after 24 h in vitro [9]. The dose of VIP (10 211 M) was chosen based on previous studies where we found that this was the most effective in a range of concentrations from 10 27 to 10 213 M. In contrast, in an earlier report in which we studied the dose-related effect of VIP upon SS secretion, 10 27 M was the most effective dose [9]. There are some differences in the culture conditions that could account for this discrepancy. In the present study we used defined medium instead of serum-free medium, cells were in vitro 5–6 days before the addition of VIP, whereas in our previous study experiments were conducted after 10 DIV, and the time of exposure to exogenous VIP was 2 and 5 days instead of 1 day. We have also observed that 10 211 M VIP is more effective than 10 27 VIP in eliciting proliferation of neuroblastoma cell lines (NB-69) (Abstract, SFN 1997). Stimulation of different receptor populations or receptor de-

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sensitization due to down regulation could account for these effects. Thus, it has been reported that stimulation of HEK293 cells stably expressing the hVIP2 / PACAP.R with increasing concentrations of VIP elicited a reduction in the extent of cAMP production [32]. The potential role of VIP in the response of SS mRNA expression to BDNF induction was assessed by blocking locally produced VIP with specific antibodies which resulted in a dramatic reduction of BDNF-induced SS mRNA. These findings suggest that VIP mediates in the induction of SS by BDNF. Supporting this hypothesis is the fact that the levels of IR-VIP were higher in the BDNF than in the VIP-treated cultures (Table 1). This suggests that after 5 days in culture exogenous VIP is degraded, whereas BDNF-induced VIP remains and exerts a biological effect. When BDNF was added in the presence of BDNF-Ab, the blockade was more effective than that obtained with VIP-Ab. This further supports that some BDNF-induced SS mRNA is mediated by the BDNFinduced VIP. As the blockade of BDNF action by the VIP-Ab was not complete, and 50% of the interneurons express the TrkB receptor, it could be argued that some of the effects of BDNF on SS mRNA are exerted by a direct interaction of BDNF with the somatostatinergic cells. After 2 days of incubation with BDNF and VIP simultaneously, we did not observe an increase of SS mRNA above that obtained with BDNF alone. However after 5 days, analysis of SS gene expression revealed an increase of accumulation, greater than that observed in cultures treated with BDNF and VIP separately. These results suggest the possibility that both factors could synergize to result in an increased and prolonged activation of SS gene expression. Interestingly, this period of 5 days in culture was coincident with the maximum effect of BDNF on VIP. Therefore, the high levels of VIP peptide in culture might promote the increment on SS mRNA directly or indirectly through BDNF synthesis as has been previously described [39]. At 5 days, single VIP addition alone failed to increase SS mRNA but its combination with BDNF exerted the effect on top of the BDNF alone. As IR-VIP concentration was higher, although not significantly different, in the BDNF-VIP than in the BDNF-treated cultures (Table 1), this difference might contribute to potentiate the effect of BDNF on SS mRNA. The existence of a cAMP-dependent regulation of neurotrophin signaling should also be considered, as VIP-induced cAMP levels might promote BDNF peptidergic action. Recently it has been shown that cAMP not only acts in a permissive capacity but also modulates the potency of BDNF action at the synapse [3]. Another group [33] has demonstrated that cAMP elevation rapidly recruits TrkB to the plasma membrane by translocation from intracellular stores. Thus we would expect elevation of VIP levels by BDNF to lead to the accumulation of cAMP and result in the up-regulation of TrkB. In summary, these findings demonstrate for the first time the action of BDNF on VIP peptide which might play a

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prominent role as a mediator of BDNF actions in the developing brain.

Acknowledgements We thank Dr. Richard Goodman for the rat SS cDNA and VIP cDNA clones and Amgem Inc. for the donation of the rhBDNF. We also thank Costanza Navarro for technical assistance and Mary Harper for the preparation of the manuscript. This work was supported by grants from FIS 99 / 0204 and 98 / 0343, CAM 08.6 / 0002 / 1997, DGCYT ´ Arebi. PB 98-1629-C02-01 and Fundacion

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