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Endocrinology Vol. 143, No. 4 1337-1345
Copyright © 2002 by The Endocrine Society

NEUROENDOCRINOLOGY

Evidence that Brain-Derived Neurotrophic Factor Acts as an Autocrine Factor on Pituitary Melanotrope Cells of Xenopus laevis

Bianca M. R. Kramer, Peter M. J. M. Cruijsen, Debbie T. W. M. Ouwens, Marcel W. Coolen, Gerard J. M. Martens, Eric W. Roubos and Bruce G. Jenks

Departments of Cellular Animal Physiology (B.M.R.K., P.M.J.M.C., D.T.W.M.O., E.W.R., B.G.J.) and Molecular Animal Physiology (M.W.C., G.J.M.M.), Nijmegen Institute for Neurosciences and Institute of Cellular Signalling, University of Nijmegen, Nijmegen 6525 ED, The Netherlands

Address all correspondence and requests for reprints to: Bruce G. Jenks, Department of Cellular Animal Physiology, Nijmegen Institute for Neurosciences, University of Nijmegen, Toernooiveld 1, Nijmegen 6525 ED, The Netherlands. E-mail: . jenks{at}sci.kun.nl


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
 References
 
We have investigated the physiological regulation and functional significance of brain-derived neurotrophic factor (BDNF) in the endocrine melanotrope cells of the pituitary pars intermedia of the amphibian Xenopus laevis, which can adapt its skin color to the light intensity of its environment. In black-adapted animals, melanotrope cells produce and release {alpha}-melanophore-stimulating hormone ({alpha}-MSH). In white-adapted animals, the activity of melanotrope cells is inhibited by neuronal input. Using Western blotting and immunocytochemistry at the light and electron microscopical level, we have detected both the BDNF precursor and the mature BDNF protein in Xenopus melanotrope cells. In situ hybridization and RT-PCR revealed the presence of BDNF mRNA in the pituitary pars intermedia, indicating that BDNF is synthesized in the melanotropes. Real-time quantitative RT-PCR showed that levels of BDNF mRNA in melanotrope cells are about 25 times higher in black- than in white-adapted animals. Although there is no difference in the amount of stored mature BDNF, the amount of BDNF precursor protein is 3.5 times higher in melanotropes of black-adapted animals than in those of white-adapted animals. These data indicate that BDNF mRNA expression and BDNF biosynthesis are up-regulated in active melanotrope cells. Because immunoelectron microscopy showed that BDNF is located in melanotrope secretory granules, BDNF is probably coreleased with {alpha}-MSH via the regulated secretory pathway. Superfusion and 3H-amino acid incorporation studies demonstrated that BDNF stimulates the release of {alpha}-MSH and the biosynthesis of its precursor protein, POMC. Our results provide evidence that BDNF regulates the activity of Xenopus melanotrope cells in an autocrine fashion.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Conclusions
 References
 
IT IS WELL established that brain-derived neurotrophic factor (BDNF), apart from its classical role in promoting survival and differentiation of neurons, is involved in the modification of neurotransmission and synaptic plasticity in the central and peripheral nervous system (1, 2, 3). However, its function in the pituitary gland is less clear. In the rat pituitary, BDNF is found in fibers in the posterior lobe, whereas BDNF and BDNF mRNA have been demonstrated in melanotrope cells of the intermediate lobe and in TSH-containing cells of the anterior lobe (4, 5, 6). It has been suggested that BDNF in the anterior lobe is involved in the control of thyroid function and in the regulation of the hypothalamic-pituitary-adrenal axis (4, 5).

We have investigated the physiological regulation and functional significance of BDNF in the endocrine melanotrope cells of the pituitary pars intermedia of Xenopus laevis. The background adaptation process of this amphibian provides the opportunity to manipulate the activity of the melanotrope cells as well as their regulatory neuronal input in vivo in a physiological way. In animals adapted to a black background, the melanotrope cells produce and release {alpha}-melanophore-stimulating hormone ({alpha}-MSH), which causes pigment dispersion in skin melanophores, giving the animal a black appearance. In white-adapted animals, the activity of the melanotrope cells is inhibited by neurons in the hypothalamic suprachiasmatic nucleus [suprachiasmatic melanotrope-inhibiting neurons (SMINs)], which make synaptic contacts with the melanotropes. SMINs contain dopamine, NPY, and {gamma}-aminobutyric acid, are more active in white- than in black-adapted animals, and have inhibitory synapses that are larger and more numerous in animals adapted to a white background (7, 8).

In this study, the presence and regulation of BDNF and BDNF mRNA in the pars intermedia have been investigated using Western blotting, immunocytochemistry at the light and electron microscopical level, real-time quantitative RT-PCR, and in situ hybridization. In addition, the role of BDNF in the regulation of {alpha}-MSH release and biosynthesis of POMC, the precursor protein of {alpha}-MSH, was studied using superfusion and 3H-amino acid incorporation. We demonstrated an up-regulation of BDNF mRNA expression and BDNF biosynthesis in melanotrope cells of black-adapted animals and provided evidence that BDNF has an autocrine role in regulating the activity of the melanotrope cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Conclusions
 References
 
Animals
Young adult (aged 8 months) specimens of the aquatic toad Xenopus laevis, weighing 28–32 g, were reared in our laboratory under standard conditions. They were kept in filtered tap water of 22 C under constant illumination and fed beef heart and trout pellets (Trouvit, Trouw, Putten, The Netherlands). Full background adaptation was achieved by keeping the animals on a white or black background for 3 wk. Experimental procedures were performed at 20 C, unless stated otherwise. All experiments were carried out under the guidelines of the Dutch law concerning animal welfare.

Cell and tissue preparation
Animals were anesthetized by immersion in an ice-cold solution of 0.1% tricaine methane sulfonate (Sigma, St. Louis, MO). For immunoelectron microscopy, RT-PCR, Western blotting, and biosynthesis experiments, animals were decapitated and brains and pituitary neurointermediate lobes (NILs) were rapidly dissected. Dissociated cells for Western blotting and superfusion experiments were obtained as described previously (9), with minor modifications. In brief, animals were transcardially perfused with 10 ml Ringer’s solution [112 mM NaCl, 2 mM KaCl, 2 mM CaCl2, 15 mM HEPES (Calbiochem, La Jolla, CA), 10 mM glucose, and 0.025% tricaine methane sulfonate pH 7.4)] to remove blood cells. After decapitation, NILs were rapidly dissected and pooled in Xenopus Leibovitz medium (XL15) consisting of 76% Leibovitz medium (Life Technologies, Inc., Paisley, UK), 1% kanamycin, 1% antibiotic/antimyotic solution (Life Technologies, Inc.), 2 mM CaCl2, and 10 mM glucose (pH 7.4). Subsequently, cells were dissociated by incubation in 1% collagenase type V (Sigma) and 2% dispase II (Roche, Basel, Switzerland) in XL15 for 1 h at 21 C, followed by gentle trituration through a siliconized Pasteur’s pipet. The suspension was filtered through a nylon gauze (pore size 150 µm) to remove undissociated tissue. Dissociated cells were washed three times with XL15 and collected by centrifugation at 50 g. For immunocytochemistry and in situ hybridization, animals were transcardially perfused with ice-cold 0.6% NaCl solution for 5 min, followed by ice-cold Bouin’s fixative for 15 min. After dissection, brains including the pituitary gland were postfixed in Bouin’s fixative for 2 h. For immunocytochemistry, brains were cryoprotected in 30% sucrose in 0.1 M sodium phosphate buffer (PB; pH 7.4) for 16 h at 4 C and subsequently frozen in Tissue-Tek (Sakura, Tokyo, Japan). Coronal cryostat sections (20 µm) were mounted on poly-L-lysine-coated slides. Slides were dried at 37 C for 16 h before further processing. For in situ hybridization, brains were dehydrated in a graded series of ethanol and xylene and embedded in paraffin. Coronal sections of 7 µm were mounted on poly-L-lysine coated slides, which were dried for 16 h at 37 C before further processing.

SDS-PAGE and Western blotting
Homogenates of whole brains, NILs, and dissociated cells were made in sample buffer (62.5 mM Tris-HCl, 12.5% glycerol, 1.25% SDS, 0.0125% bromophenol blue, and 2.5% ß-mercaptoethanol). Whole brains (not including the pituitary gland) were crushed in a glass homogenizer in 1 ml sample buffer each on ice and homogenized by passage through a 23-gauge syringe needle. NILs were homogenized in 50 µl sample buffer each by keeping them on ice for 3–4 h. Dissociated cells from six NILs were resuspended in 25-µl sample buffer on ice. All samples were lysed by boiling. Whole-brain samples were diluted 1:8 in sample buffer. Equal volumes (25 µl) of samples were separated on a 12.5% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane (0.45 µm, Schleicher \|[amp ]\| Schuell, Inc., Dassel, Germany) using a miniprotean II cell system (Bio-Rad Laboratories, Inc., Hemel Hempstead, UK). Molecular mass markers (Benchmark prestained protein ladder, 10–200 kDa, Life Technologies, Inc.) were run alongside the samples to be analyzed. For immunodetection, blots were washed three times in 0.1 M Tris-buffered saline (TBS) (pH 7.5) containing 0.2% Tween-20 (Sigma) (TBSTw) and 1% skimmed milk (wash buffer). To prevent aspecific binding, blots were incubated in TBSTw and 5% skimmed milk (block buffer) for 1 h. Subsequently, blots were incubated in primary antiserum (rabbit polyclonal IgG against the 20 N-terminal amino acids of the mature BDNF protein of human origin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), diluted 1:100 in blocking buffer for 16 h. After three rinses in wash buffer, blots were incubated in horseradish peroxidase (HRP)-conjugated goat antirabbit IgG (1:1000, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) for 1 h. After three rinses in TBS, proteins were visualized by incubating blots in Lumi-LightPLUS Western blotting substrate (Roche), followed by exposure to x-ray film (Eastman Kodak Co., Rochester, NY). Signal was quantified with a GS 700 densitometer (Bio-Rad Laboratories, Inc.) using Molecular Analyst software (Bio-Rad Laboratories, Inc.). Specificity of the primary antiserum was tested on a sample of human recombinant BDNF (2 ng, Santa Cruz Biotechnology) and by preabsorption of the antiserum with antibody-specific blocking peptide (Santa Cruz Biotechnology).

Immunocytochemistry
To quench endogenous peroxidases, sections were incubated with 1% H2O2 in TBS for 15 min. After rinsing in TBS containing TBSTw (Sigma), sections were incubated in 0.5% TSA blocking reagent (NEN Life Science Products, Boston, MA) in TBS for 20 min to prevent aspecific binding. Subsequently, they were incubated in rabbit anti-BDNF serum (1:50, Santa Cruz Biotechnology) in blocking reagent in TBS for 16 h, rinsed in TBSTw and incubated in HRP-conjugated donkey antirabbit IgG (1:100, Jackson ImmunoResearch Laboratories, Inc.) in TBSTw for 30 min. After rinsing in TBSTw, sections were incubated in fluorescein-conjugated Tyramide solution (1:50 in Amplification Diluent, NEN Life Science Products) for 10 min, rinsed in TBS, and coverslipped in FluorSave (Calbiochem). Sections were examined on a Leica Corp. DM-RB/E fluorescence microscope (Leica, Solms, Germany). Specificity of the staining was tested by omitting the first antiserum and preabsorbing the first antiserum with antibody-specific blocking peptide (Santa Cruz Biotechnology).

Immunoelectron microscopy
Dissected NILs were fixed in 1% glutaraldehyde in PB for 16 h. They were subsequently treated with 0.2% borohydride and 0.4% glycine in PB for 15 min and cryoprotected by immersion in a graded series of glycerol. Lobes were rapidly frozen in liquid propane (-180 C) and placed in a precooled chamber (-90 C) of a quick-freezing apparatus (Reichert-Jung, Vienna, Austria). The tissue was freeze-substituted by methanol containing 0.5% uranyl acetate and embedded in Lowicryl HM20 resin (Bio-Rad Laboratories, Inc.). Ultrathin gold sections were collected on Formvar-coated 150-mesh nickel grids. After treatment with 50 mM glycine in 0.01 M sodium PBS (pH 7.4) for 10 min, rinsing in 0.2% gelatin and 0.5% BSA (ICM Biomedicals, Costa Mesa, CA) in PB and incubation in 1% BSA in PBS for 10 min, sections were immersed in rabbit anti-BDNF serum (1:250, Santa Cruz Biotechnology) in 1% BSA in PBS for 16 h, rinsed in 0.2% gelatin and 0.5% BSA in PB, and incubated in 10 nm gold-conjugated goat antirabbit IgG (Aurion, Wageningen, The Netherlands) for 2 h. Subsequently, sections were postfixed in 1% glutaraldehyde in PB for 10 min and contrasted in 2% uranyl acetate and lead citrate. Sections were examined with a JEOL TEM 1010 electron microscope (JEOL, Tokyo, Japan). Specificity of the staining was tested by omitting the first antiserum.

In situ hybridization
A Xenopus-specific BDNF cDNA clone that codes for 10 amino acids (aa) of the terminal precursor region and 119 aa of the mature BDNF (acc. number X61477, 10), subcloned in the HincII site of the PBS plasmid, was used to generate mRNA probes. After linearization of the plasmid with HindIII or EcoRI, 11-UTP digoxigenin (DIG)-labeled antisense and sense probes were prepared as run-off transcripts using T7and T3 DNA polymerase (Roche).

Sections were deparaffinated in xylene and rehydrated in a graded series of ethanol. Tissue penetration was enhanced by incubation in 0.1% pepsin in 0.2 M HCl for 15 min at 37 C, followed by fixation in 4% paraformaldehyde in PBS for 5 min and incubation in 1% HONH3Cl for 15 min. Sections were subsequently dehydrated in ethanol and air dried. Hybridization took place for 16 h at 55 C in hybridization buffer (10% sodium dextran sulfate, 50% formamide, 4x salt and sodium citrate (SSC), 1x Denhardt’s, and 200 µg/ml yeast tRNA; 1x SSC = 0.15 M NaCl and 0.015 M sodium citrate) with 500 ng/ml antisense DIG-labeled BDNF mRNA probe. After stringency washes in 2x SSC, 1x SSC, 0.5x SSC for 30 min and 0.1x SSC for 30 min at 37 C, sections were rinsed for 10 min in TBS, blocked in 1% BSA (ICM Biomedicals) and 2% normal goat serum in TBS (blocking solution) for 30 min, and incubated in alkaline phosphatase (AP)-conjugated sheep anti-DIG Fab fragments (1:500, Roche) in blocking solution for 16 h at 4 C. After three washes of 10 min in TBS and one wash of 5 min in AP buffer (100 mM Tris, 100 mM NaCl; pH 9.5), sections were stained in 350 µg/ml 4-nitro blue tetrazolium chloride and 175 µg/ml 5-bromo-4-chloro-3-indolyl-phosphate (Roche) in AP buffer until color development was sufficient. Specificity was checked by hybridization with the sense BDNF-mRNA probe.

Real-time quantitative RT-PCR
Freshly dissected NILs were individually collected in 500-µl ice-cold TRIzol (Life Technologies, Inc.) and homogenized by sonication. After chloroform extraction and isopropyl alcohol precipitation, RNA was redissolved in 15 µl RNase-free H2O. Total RNA was measured with a biophotometer (Vaudaux-Eppendorf AG, Basel, Switzerland). First-strand cDNA synthesis was performed using 11 µl RNA and 1 µl pd(N)6 (random primers, 5 mU/µl, Roche) at 70 C for 10 min, followed by double-strand synthesis in 20 µl strand buffer (Life Technologies, Inc.) with 10 mM DTT, 20 U RNAsin (Promega Corp., Madison, WI), 0.5 mM dNTPs (Roche), and 100 U reverse transcriptase (SuperScript II, Life Technologies, Inc.) at 37 C for 60–90 min. For controls, reverse transcriptase was omitted from the reaction mixture. Samples were diluted five times in RNase-free H2O and subjected to real-time quantitative RT-PCR on a 5700 GeneAmp PCR system (PE Applied Biosystems, Wellesley, MA) as follows: 5 µl template cDNA was added to 20 µl SYBR green buffer (PE Applied Biosystems) with 3 mM MgCl2, 0.2 mM dNTPs (PE Applied Biosystems), 0.6 µM of each primer, and 0.625 U AmpliTaq gold (PE Applied Biosystems). PCR conditions were 95 C for 10 min followed by 40 reaction cycles of 95 C for 15 sec and 60 C for 1 min each. Primers were designed using Vector NTI Suite (InforMax, Bethesda, MD) and PrimerExpress (PE Applied Biosystems) software based on the published Xenopus mRNA sequence (account number X61477, 10) for BDNF and the Xenopus cDNA sequence (account number U41753) for glyceraldehyde 3-phosphate dehydrogenase (GADPH). The following primer pairs were used: BDNF, forward 5'-CGTGGAGAGCTGAGTGTGTGTGAC-3' and reverse 5'-GTTGGCCTTTGGATACTGGGACTT-3' (product size 127 bp); GADPH, forward 5'-GCCGTGTATGTGGTGGAATCT-3' and reverse 5'-AAGTTGTCGTTGATGACCTTTGC-3' (product size 230 bp). For each reaction, the cycle threshold (Ct) was determined, i.e. the cycle number at which fluorescence was detected above an arbitrary threshold (0.8). At this threshold, Ct values are within the exponential phase of the amplification. To estimate the relative amount of BDNF mRNA in NILs from black- vs. white-adapted animals, Ct values were normalized to those of the internal standard (GADPH) and compared. After PCR, the reaction products were run on a 2.5% agarose gel and visualized with ethidium bromide to check the length of the amplified DNA.

Superfusion
Dissociated cells from 1.5 NILs were resuspended in 50 µl lysine-free XL15 with 10% dialyzed FCS (Life Technologies, Inc.) containing 62.5 µCi 3H-lysine (80 Ci/mM; Amersham Pharmacia Biotech, Buckinghamshire, UK). The cell suspension was plated on a poly-L-lysine coated coverslip and allowed to attach for 1 h at 21 C, after which 50 µl lysine-free XL15 with 20% dialyzed FCS was added, and the cells were incubated for 16 h at 21 C. Then 2 ml lysine-free XL15 with 10% FCS was added, and cells were incubated for another 24 h at 21 C. Subsequently, coverslips were rinsed three times with Ringer’s solution and placed individually in 4-well culture dishes (Nunclon, Roskilde, Denmark) in Ringer’s solution. Cells were superfused with Ringer’s solution at a rate of 100 µl/min. At specific time points, BDNF, sauvagine (Bachem, Bubendorf, Switzerland), and apomorphine (Sigma) were added to the superfusion medium, following the protocol given in Results. Fractions of 2 min were collected, to which 200 µl of scintillation liquid (Optiphase Supermix, Wallac, Inc., Loughborough, UK) was added. The amount of radiolabeled peptides in each fraction was determined with a scintillation counter (Wallac, Inc.). The average amount of radioactivity in the three fractions immediately preceding the first pulse of BDNF was set at 100%, and the amount of radioactivity of all other fractions was expressed relative to this value. It has previously been shown that, following this protocol, about 30% of the radioactivity in the superfusate is unincorporated 3H-lysine, and approximately 65% reflects the secretion of radiolabeled {alpha}-MSH and other POMC-derived peptides (11).

POMC biosynthesis
After dissection, NILs were collected in XL15. After rinsing several times in XL15, they were incubated individually in 4-well culture dishes, each containing 0.5 ml incubation medium for 3 d at 21 C. For the control lobes, incubation medium consisted of XL15 with 10% FCS. For the experimental groups, 10-6 M NPY was added to the incubation medium. Media were refreshed daily. On d 3, 100 ng/ml BDNF was added to the incubation medium of one experimental group for 4 h. Subsequently, all lobes were pulse labeled for 15 min in 10 µl Ringer’s solution containing 1 mCi/ml 3H-lysine. Lobes were washed three times in Ringer’s solution and lysed by boiling in sample buffer for 5 min before electrophoresis. Proteins were separated on a 12% SDS-polyacrylamide gel using 20% of each lobe extract. Gels were fixed in 40% methanol and 10% acetic acid, saturated in 100% dimethylsulfoxide, and treated with 2% 2,5-diphenyloxazol in 100% dimethylsulfoxide for visualization of radioactivity, followed by exposure to x-ray film (Eastman Kodak Co.). Signal was quantified with a GS 700 densitometer using Molecular Analyst software (Bio-Rad Laboratories, Inc.).

Statistics
Data from Western blotting, real-time quantitative RT-PCR, and biosynthesis experiments were analyzed with a one-way ANOVA (12) followed by a post hoc comparison of means using Duncan’s multiple range test (Ref. 13 ; {alpha} = 5%).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Conclusions
 References
 
Presence of BDNF in Xenopus melanotrope cells
Using Western blotting, the primary antiserum, which recognized 14-kDa human recombinant BDNF, revealed the presence of 14-kDa and 35-kDa immunoreactive bands in homogenates of whole brain (n = 8), NILs (n = 8), and dissociated melanotrope cells (n = 4) (Fig. 1Go). Following preadsorption of the primary antiserum with antibody-specific blocking peptide, no signal was observed in homogenates of either whole brain or NILs.



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  		Figure 1. Western blots of human recombinant BDNF (hrBDNF), Xenopus whole brain (Brain), neurointermediate lobe (NIL), and dissociated melanotrope cells (Cells) showing 14 kD and 35 kD BDNF-immunoreactive bands (arrows). Molecular mass markers are indicated on the left.

 
Immunocytochemistry of the pars intermedia showed the presence of BDNF in melanotrope cells (n = 4, Fig. 2AGo). The specificity of this staining was confirmed by the absence of positive staining when the antibody was preadsorbed with antibody-specific blocking peptide. Immunoelectron microscopy revealed many gold particles in the secretory granules of melanotrope cells, but the rest of the cytoplasm and the nucleus were devoid of staining. Also, no immunoreactivity was observed in the glial-like folliculostellate cells, nerve fibers, and varicosities (n = 3, Fig. 3Go).



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  		Figure 2. Immunofluorescence of BDNF (A) and in situ hybridization of BDNF mRNA (B) in black-adapted Xenopus pituitary. Pd, Pars distalis; pi, pars intermedia; pn, pars nervosa. Scale bar, 50 µm.

 


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  		Figure 3. Immunoelectron microscopy of BDNF in the pars intermedia of black- (A) and white-adapted (B) Xenopus. A, Immunogold particles occur exclusively in the secretory granules (g) of the melanotrope cells (M). FS, Folliculostellate cell; n, nucleus. Scale bar, 300 nm. Inset, Immunoreactive secretory granule. Scale bar, 75 nm. B, Nerve terminals containing large electron-dense (v1) and small electron-lucent (v2) vesicles are free of immunogold particles. M, Melanotrope cell. Inset, Low-contrast image of dense vesicles. Note presence of a few gold particles outside nerve terminal (arrow).Scale bar, 300 nm.

 
The presence of BDNF mRNA in NIL tissue was demonstrated by RT-PCR, which resulted in a single PCR fragment with the expected length of 127 bp (n = 6, Fig. 4Go). Using in situ hybridization, expression of BDNF mRNA was found throughout the pars intermedia (n = 4, Fig. 2BGo). With the sense mRNA probe, no positive signal was detected.



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  		Figure 4. Agarose gel electrophoresis of reaction product of RT-PCR on total RNA from NILs of black-adapted animals using primers for BDNF, showing one band of 127 bp. Molecular mass markers are indicated on the left.

 
Regulation of BDNF in melanotrope cells
Real-time quantitative RT-PCR showed an increase of BDNF mRNA in NILs of black-adapted animals (n = 3), compared with white-adapted animals (n = 3, Fig. 5Go). Ct values for BDNF were lower in black- than in white-adapted animals (26.2 ± 0.1 and 31.6 ± 0.6, respectively), but Ct values for the housekeeping gene product GADPH did not differ significantly (25.4 ± 0.6 and 26.1 ± 0.5). The difference between normalized Ct values for BDNF in black- and white-adapted animals was 4.7. Because an increase in Ct value of 1.0 corresponds to a 2-fold increase in amount of mRNA present, this increase of 4.7 represents an approximately 25-fold increase in the amount of BDNF mRNA in NILs of black- vs. white-adapted animals. Using Western blotting, the amount of BDNF precursor protein in NILs was found to be higher (OD x3.5, Figs. 6Go and 7Go) in black- (n = 3) than in white- (n = 3) adapted animals. The amount of mature BDNF did not differ significantly between adaptation states.



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  		Figure 5. Real-time quantitative RT-PCR on total RNA from individual NILs of black- (B) and white-adapted (W) animals using primers for BDNF. Ct, Cycle threshold, i.e. cycle number at which fluorescence was detected above an arbitrary threshold (0.8).

 


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  		Figure 6. Western blot of NIL from black- (B) and white-adapted (W) animal showing BDNF precursor protein (35 kDa) and BDNF mature protein (14 kDa).

 


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  		Figure 7. OD of immunoreactive bands of BDNF precursor protein (35 kDa) and mature protein (14 kDa) on Western blot of NILs of black- (B) and white-adapted (W) animals. Values are expressed in arbitrary units (a.u.) as means ± SEM. Asterisk indicates significant difference (P < 0.002).

 
Effect of BDNF on {alpha}-MSH release
BDNF (10 ng/ml) had a clear stimulatory action on {alpha}-MSH release from dissociated melanotrope cells (n = 4, Fig. 8Go). After application of BDNF, it took about 10 min for the {alpha}-MSH release to rise. The effect of a 10-min BDNF pulse lasted for about 20 min.



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  		Figure 8. Effect of BDNF, sauvagine (sauv), and apomorphin (apo) on the release of radiolabeled {alpha}-MSH and other POMC-derived peptides from dissociated melanotrope cells from black-adapted animals. Averages - SEM of four superfusion experiments are shown.

 
A second pulse of 50 ng/ml BDNF was no longer effective in stimulating {alpha}-MSH release. Following BDNF pulses, a pulse of the stimulatory agent sauvagine (10-8 M) stimulated {alpha}-MSH release, but a pulse of the dopamine agonist apomorphine (10-8 M) inhibited {alpha}-MSH release. This showed that the melanotropes were still viable and able to respond to stimulatory and inhibitory factors.

Effect of BDNF on POMC biosynthesis
Following 3H-amino acid incorporation and SDS-PAGE, two bands of heavily radiolabeled protein were observed in extracts of NILs from black-adapted animals (Fig. 9AGo). These bands, of 38.2 and 37.3 kDa, represent the protein products of the two POMC genes, POMC-A and POMC-B (14) These proteins have been extensively characterized in Xenopus NIL extracts (15). In control lobes (n = 5), the density of these bands was very high, reflecting a high rate of POMC biosynthesis in melanotropes of black-adapted animals. To be able to show a possible stimulatory effect of BDNF on POMC biosynthesis, the high rate of this biosynthesis was first inhibited by incubation of the lobes with 10-6 M NPY (16). This resulted in a 60% decrease in POMC biosynthesis, compared with controls (n = 5). Incubation of NPY-treated lobes with 100 ng/ml BDNF resulted in a 44% increase in POMC biosynthesis (n = 4, Fig. 9Go).



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  		Figure 9. Autoradiography (A) and optical density (B) of 3H-amino acid incorporation in POMC in extracts of NILs of black-adapted animals (controls, CON), after incubation with NPY or NPY + BDNF. In A, two bands are visible, representing the protein products of the two POMC genes in Xenopus. In B, values are expressed as means ± SEM. Asterisks indicate significant differences (*, P < 0.02; **, P < 0.0002).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Conclusions
 References
 
Expression of BDNF in melanotrope cells
Using Western blotting, we observed BDNF immunoreactive bands of 14 and 35 kDa in Xenopus brain and pars intermedia. We concluded that these bands represent mature and precursor BDNF, respectively, because their molecular masses match those observed for mature and precursor BDNF in the rat (17), and human recombinant BDNF also yields a band of 14 kDa. Xenopus, rat, and human mature BDNF all consist of approximately 120 aa and show strong sequence homology. Comparative sequencing studies reveal that mature and precursor BDNF are strongly conserved among vertebrate species (10, 18, 19).

The presence of BDNF in the pars intermedia was confirmed by immunocytochemistry. At the ultrastructural level, BDNF was shown to occur exclusively in the melanotrope cells and not in glial-like folliculostellate cells and nerve terminals in the pars intermedia. The demonstration of BDNF mRNA in melanotropes by RT-PCR and in situ hybridization showed that BDNF is indeed produced by these cells and not taken up from neurons projecting to the pars intermedia, a phenomenon that has been shown to occur in the central nervous system of birds and mammals (20, 21, 22). Our findings on the amphibian Xenopus confirm earlier observations in the mammalian pituitary (4, 5, 6) and therefore suggest that the presence of BDNF in melanotrope cells of the pituitary is conserved throughout evolution. Immunoelectron microscopy showed that BDNF occurs in the secretory granules of melanotrope cells. This strongly suggests that BDNF coexists in these granules with POMC and {alpha}-MSH (23, 24, 25).

We observed higher levels of BDNF mRNA and BDNF precursor protein in pars intermedia tissue of black- than white-adapted animals, suggesting that BDNF mRNA expression and biosynthesis are up-regulated in active melanotrope cells. This phenomenon of precursor up-regulation has also been observed for POMC, the precursor protein of {alpha}-MSH. Melanotropes of black-adapted animals, which have high levels of POMC mRNA and high levels of POMC biosynthesis (26, 27), also show higher amounts of POMC protein, compared with white-adapted animals (28). Remarkably, there is no difference in the level of mature BDNF between melanotropes of black- and white-adapted animals. Possibly, this reflects release of mature BDNF from melanotropes in black-adapted animals, and in white-adapted animals the protein would be stored.

Previously, indications for regulated release of BDNF were obtained from studies by others on cells kept in vitro. For example, in hippocampal and cortical neurons, as well as in PC12 and AtT20 neuroendocrine cells, BDNF is located in the trans-Golgi network, sorted into dense-cored vesicles (29, 30, 31) and released on depolarization through the regulated secretory pathway (31, 32, 33, 34). On the basis of our in vivo studies, namely comparing two physiological conditions (black- vs. white-adapted animals), we concluded that BDNF is released in a physiologically controlled way via the regulated secretory pathway. This conclusion is based on the fact that BDNF can be assumed to coexist with {alpha}-MSH in secretory granules in the melanotrope cells. The release of the contents of these secretory granules is under physiological control of various neurotransmitters and neuropeptides. Secretion is stimulated in black-adapted animals and inhibited in white-adapted animals (7, 8). This implies that the release of BDNF from Xenopus melanotropes would be physiologically regulated.

Our results from Western blotting and real-time quantitative RT-PCR indicates that not only the release but also the production of BDNF is under physiological control. The melanotropes of white-adapted animals receive enhanced inhibitory input from the SMINs. Whether the background-induced decrease in BDNF mRNA and BDNF precursor protein in melanotropes of white-adapted animals is mediated by dopamine, {gamma}-aminobutyric acid, and/or NPY, the three transmitters released from the SMINs, remains to be investigated. In the rat, it has been shown that dopamine has a regulatory influence on the amount of BDNF present in the melanotrope cells (35), but it is not known whether this control concerns the biosynthesis or secretion of BDNF.

Action of BDNF on melanotrope cells
Our studies revealed that BDNF has a stimulatory action on the melanotrope cells, increasing both {alpha}-MSH release and POMC biosynthesis. The low effective concentration (10 ng/ml) at which BDNF stimulates the basal release of {alpha}-MSH from dissociated melanotrope cells indicates that BDNF acts via its high-affinity receptor tropomyosin kinase B (TrkB) (Kd = 1.7 x 10-11 M, or 0.5 ng/ml) rather than via the low-affinity neurotrophin receptor p75 (Kd = 1.5 x 10-9 M, or 35 ng/ml; Ref. 36). This stimulatory effect on {alpha}-MSH release is transient, lasting for 15–20 min after the BDNF pulse. This is another indication for its binding to TrkB because this receptor has a much slower dissociation rate than p75 (t1/2 = 10 min/3 sec, respectively, 37). The presence of TrkB on melanotrope cells has been demonstrated in the adult rat (38), and p75 is detectable only during development (39, 40). A second pulse of BDNF proved to be no longer effective in stimulating {alpha}-MSH-release from Xenopus melanotropes. This might reflect a rapid down-regulation of the TrkB receptor, as has been shown for cultured cerebellar granule cells and a neuronal cell line (41). Despite this down-regulation, BDNF clearly has long-term effects on Xenopus melanotropes, indicated by the fact that BDNF stimulates POMC biosynthesis.

Apart from acting directly on the melanotrope cells, BDNF might also work presynaptically on nerve terminals innervating these cells. In the rat, TrkB immunoreactivity has been observed on nerve endings in the intermediate lobe (38). BDNF also enhances neurite outgrowth and levels of tyrosine hydroxylase in hypothalamic dopaminergic neurons that project to the intermediate lobe (42). It would be interesting to investigate whether in Xenopus BDNF plays a role in regulating the plasticity of the synaptic innervation of the melanotrope cells, in addition to its actions on the melanotrope cells themselves.


   Conclusions
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
References
 
In this study, the physiological regulation and functional significance of BDNF in the endocrine melanotrope cells of the pituitary pars intermedia of Xenopus laevis were investigated. We have demonstrated the presence of BDNF mRNA and protein in melanotrope cells. BDNF occurs in secretory granules, probably coexisting with {alpha}-MSH. BDNF mRNA expression and biosynthesis are up-regulated in active cells, and BDNF in turn stimulates the biosynthesis and release of {alpha}-MSH. On the basis of these results, we propose that BDNF is an autocrine factor regulating the activity of Xenopus melanotrope cells.


   Acknowledgments
 
The authors are grateful to R. J. C. Engels for animal care and to Dr. S. Cohen-Cory for the generous gift of Xenopus BDNF cDNA.


   Footnotes
 
This work was supported by a grant from the Space Research Organization Netherlands (SRON), which is subsidized by The Netherlands Organization for Scientific Research (NWO).

Abbreviations: aa, Amino acids; {alpha}-MSH, {alpha}-melanophore-stimulating hormone; AP, alkaline phosphatase; BDNF, brain-derived neurotrophic factor; Ct, cycle threshold; DIG, digoxigenin; GADPH, glyceraldehyde 3-phosphate dehydrogenase; HRP, horseradish peroxidase; NIL, neurointermediate lobe; PB, sodium phosphate buffer; SSC, salt and sodium citrate; SMIN, suprachiasmatic melanotrope-inhibiting neuron; TBS, Tris-buffered saline; TBSTw, 0.2% Tween-20; TrkB, tropomyosin kinase B; XL15, Xenopus Leibovitz medium.

Received August 15, 2001.

Accepted for publication December 7, 2001.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
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