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Keywords:

  • Brain-derived neurotrophic factor;
  • Neurotrophin-3;
  • Neurotrophin-4;
  • Calretinin;
  • Calbindin;
  • Cerebral cortex

Abstract

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. RESULTS
  5. Effects of BDNF on number of calbindin- and calretinin-immunoreactive neurons
  6. Effects of BDNF on levels of calbindin and calretinin mRNAs
  7. DISCUSSION
  8. Acknowledgements

Abstract: Regulation of calbindin and calretinin expression by brain-derived neurotrophic factor (BDNF) was examined in primary cultures of cortical neurons using immunocytochemistry and northern blot analysis. Here we report that regulation of calretinin expression by BDNF is in marked contrast to that of calbindin. Indeed, chronic exposure of cultured cortical neurons for 5 days to increasing concentrations of BDNF (0.1-10 ng/ml) resulted in a concentration-dependent decrease in the number of calretinin-positive neurons and a concentration-dependent increase in the number of calbindin-immunoreactive neurons. Consistent with the immunocytochemical analysis, BDNF reduced calretinin mRNA levels and up-regulated calbindin mRNA expression, providing evidence that modifications in gene expression accounted for the changes in the number of calretinin- and calbindin-containing neurons. Among other members of the neurotrophin family, neurotrophin-4 (NT-4), which also acts by activating tyrosine kinase TrkB receptors, exerted effects comparable to those of BDNF, whereas nerve growth factor (NGF) was ineffective. As for BDNF and NT-4, incubation of cortical neurons with neurotrophin-3 (NT-3) also led to a decrease in calretinin expression. However, in contrast to BDNF and NT-4, NT-3 did not affect calbindin expression. Double-labeling experiments evidenced that calretinin- and calbindin-containing neurons belong to distinct neuronal subpopulations, suggesting that BDNF and NT-4 exert opposite effects according to the neurochemical phenotype of the target cell.

Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family, which also includes nerve growth factor (NGF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5) (Lewin and Barde, 1996). BDNF binds to and activates the low-affinity p75 receptor as well as the specific tyrosine kinase receptor (Trk) B through which the neurotrophin exerts its biological functions (Barbacid, 1994; Bothwell, 1995). There is abundant in vitro and in vivo evidence showing that BDNF promotes the survival and differentiation of selective populations of neurons (Davies, 1994; Lewin and Barde, 1996). For instance, BDNF acts as a peptidergic differentiation factor for GABA-containing neurons as it stimulates the expression of specific neuropeptides such as somatostatin, substance P, neuropeptide Y, and cholecystokinin both in vitro and in vivo (Nawa et al., 1993, 1994; Croll et al., 1994). Consistent with these observations, expression of neuropeptide Y is reduced in the cerebral cortex and hippocampus of BDNF knockout mice (Jones et al., 1994). In addition to its role as a peptidergic differentiation factor, BDNF has been reported to regulate the expression of calcium-binding proteins (Ip et al., 1993; Widmer and Hefti, 1994; Marty et al., 1996; Pappas and Parnavelas, 1997). Thus, BDNF stimulates calbindin expression in cultured hippocampal and cortical neurons (Ip et al., 1993; Widmer and Hefti, 1994). Along with this evidence, the expression of calbindin as well as of parvalbumin is reduced in the cerebral cortex and hippocampus of BDNF knockout mice (Jones et al., 1994). In addition to calbindin and parvalbumin, calretinin is the third member of the EF-hand family of calcium-binding proteins that is expressed throughout the brain. Although little is known regarding their precise functional properties, these three calcium-binding proteins are thought to buffer intracellular free Ca2+ (Baimbridge et al., 1992). In the adult rat cerebral cortex, calbindin, parvalbumin, and calretinin are localized in three distinct subpopulations of GABAergic neurons (Celio, 1990; Resibois and Rogers, 1992), whereas they are expressed transiently in non-GABAergic neurons during development (Fonseca et al., 1995).

Although regulation of calbindin and parvalbumin expression by BDNF has been examined in the cerebral cortex, there has been no report on the regulation of calretinin expression by BDNF. Thus, the aim of this study was to investigate the regulation of calretinin expression by BDNF in cultures of dissociated cortical neurons and to compare this effect with that observed on the expression of calbindin and parvalbumin. To examine whether the actions of other members of the neurotrophin family were similar to those of BDNF, we have also tested the effects of NGF, NT-3, and NT-4 on the expression of calbindin and calretinin.

Results reported in this study clearly show that regulation of calretinin expression by BDNF is in marked contrast to that of calbindin. Indeed, BDNF reduces the expression of calretinin mRNA and the number of calretinin-positive neurons, although it stimulates the expression of calbindin mRNA and the number of calbindin-immunoreactive neurons. NT-4 exhibited effects similar to those of BDNF, whereas NGF did not affect the expression of these two calcium-binding proteins. It is interesting that NT-3 decreased calretinin expression, whereas it did not regulate calbindin expression.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. RESULTS
  5. Effects of BDNF on number of calbindin- and calretinin-immunoreactive neurons
  6. Effects of BDNF on levels of calbindin and calretinin mRNAs
  7. DISCUSSION
  8. Acknowledgements

Preparation of primary cultures of mouse cortical neurons

All experiments were carried out in accordance to the European Communities Council Directive regarding care and use of animals for experimental procedures.

Primary cultures of cerebral cortical neurons were prepared from 17-day-old Swiss mouse embryos as previously described (Cardinaux et al., 1997; Pellegri et al., 1998; Fiumelli et al., 1999). Cells were plated at a density of 2 × 105/cm2 on 90- × 15-mm culture plates (northern blots) or on glass coverslips (12 mm in diameter; immunocytochemistry) in Dulbecco's modified Eagle's medium supplemented with 1 mM glutamine, 7.5 mM sodium bicarbonate, 5 mM HEPES buffer (pH 7.0), 0.1 mg/ml streptomycin, and 0.06 mg/ml penicillin. A mixture of hormones and salt containing 25 μg/ml insulin, 100 μg/ml transferrin, 60 μM putrescine, 20 nM progesterone, and 30 nM sodium selenate was added to the culture medium. Cells were maintained for 6 days at 37°C in a humidified atmosphere of 95% air and 5% CO2. As previously reported (Stella et al., 1995), immunostaining of 6 days in vitro cortical cultures with cell-specific antibodies yields 94% neuron-specific enolase-immunoreactive cells and 99% neurofilament-immunoreactive cells.

Chronic treatment of cultured cortical neurons with neurotrophins

For northern blot experiments, cells were exposed daily to 0.1, 1, 3, 5, or 10 ng/ml BDNF, 5 ng/ml NGF, 5 ng/ml NT-3, or 5 ng/ml NT-4 for 5 days starting 1 day after plating. On day 6, total RNA was extracted and electrophoresed as described below.

For immunocytochemistry, cultures in separate wells were exposed daily to 0.1, 0.5, 1, 5, or 10 ng/ml BDNF, 5 ng/ml NGF, 5 ng/ml NT-3, or 5 ng/ml NT-4 for 5 days starting 1 day after plating. On day 6, cultures were fixed and subjected to immunocytochemistry.

Northern blot analysis

At the end of the stimulation, cells were washed twice with ice-cold phosphate-buffered saline (PBS; pH 7.4), and total RNA was extracted from cultured cortical neurons using the CsCl centrifugation procedure (Chirgwin et al., 1979). Total RNA (10 μg) was electrophoresed on a 1.3% agarose/2 M formaldehyde gel and transferred onto a GeneScreen nylon membrane (NEN Life Science, Zaventem, Belgium). Hybridization was performed overnight at 65°C in 50% formamide, 5× saline-sodium citrate (SSC), 50 mM Tris-HCl (pH 7.5), 0.1% sodium pyrophosphate, 1% sodium dodecyl sulfate, 0.2% polyvinylpyrrolidone, 0.2% Ficoll, 5 mM EDTA, 0.2% bovine serum albumin, and 150 μg/ml salmon sperm DNA with a 32P-antisense calbindin, calretinin, or parvalbumin riboprobe. Filters were then washed under high-stringency conditions (twice with 2× SSC containing 0.1% sodium dodecyl sulfate at 65°C for 15 min and twice with 0.1× SSC containing 0.1% sodium dodecyl sulfate at 65°C for 30 min) and apposed to autoradiography films (Hyperfilms; Amersham Pharmacia Biotech, Dübendorf, Switzerland) at -70°C with an intensifying screen. Differences in RNA gel loading and blotting were assessed by rehybridizing the filters with a 32P-antisense actin riboprobe (Martin et al., 1995; Pellegri et al., 1998). Hybridization and washing conditions for actin were identical to those described above. After autoradiography, films were densitometrically scanned, and results were quantified using the Macintosh-based National Institutes of Health Image program (version 1.61). Densitometric values for calretinin and calbindin mRNAs were normalized to corresponding actin mRNA values. Results are mean ± SEM percentages of control from three independent experiments.

32P-antisense riboprobes were generated using either T7 or SP6 RNA polymerase and [α-32P]UTP from a linearized pGEM-T vector (Promega, Zürich, Switzerland) containing the appropriate cDNA fragment. The 749-bp calbindin cDNA fragment was obtained by RT and PCR amplification of total RNA from primary cultures of mouse cortical neurons with a set of oligonucleotide primers (5′ -ATGGCAGAATCCCACCTGCA-3′ and 5′ -GTTCGGTACAGCTTCCCTCC-3′) located at 286-305 and 1,015-1,034 bp, respectively, in the coding region of the rat calbindin cDNA sequence (Hunziker and Schrickel, 1988). The 490-bp calretinin cDNA fragment was obtained by RT and PCR amplification of total RNA from cultured cortical neurons with a set of oligonucleotide primers (5′ -TGCTTCAGGCAGCACGTGGG-3′ and 5′ -CAATCTCCAGGTCCTTTCTG-3′) located at 59-78 and 529-548 bp, respectively, in the coding region of the mouse calretinin cDNA sequence (Ellis and Rogers, 1993). The 333-bp parvalbumin cDNA fragment was obtained by RT and PCR amplification of total RNA from adult mouse brain with a set of oligonucleotide primers (5′ -ATGTCGATGACAGACGTGCT-3′ and 5′ -TTACGTTTCAGCCACCAGAG-3′) located at 1-20 and 314-333 bp, respectively, in the coding region of the mouse parvalbumin cDNA sequence (Zuhlke et al., 1989).

The identity of the amplified calbindin, calretinin, and parvalbumin cDNA fragments was confirmed by sequencing using an automated DNA sequencer (ALF DNA Analysis System; Pharmacia, Uppsala, Sweden).

Immunocytochemistry

Diaminobenzidine labeling. At the end of the incubation period, cultures were fixed for 25 min in 4% paraformaldehyde (except for GABA immunostaining, where a mixture of 4% paraformaldehyde and 1% glutaraldehyde was used). Depending on the primary antibody tested, cultures were preincubated for 1 h in PBS with 0.2% Triton X-100 (PBS-T) containing either 4% normal goat serum (for polyclonal antibodies) or 4% normal horse serum (for monoclonal antibodies). Cultures were then incubated overnight at room temperature in a solution containing the primary antibody diluted in PBS-T containing 4% normal goat or horse serum. The antibodies used were the following: rabbit anti-GABA (Sigma), 1:10,000; rabbit anti-calretinin (Swant, Switzerland), 1:5,000; mouse monoclonal anti-calbindin (Swant), 1:2,000; and mouse monoclonal parvalbumin (Swant), 1:2,000. The next day, cultures were rinsed in PBS-T and incubated for 30 min in the presence of biotinylated goat anti-rabbit IgG or biotinylated horse anti-mouse IgG (Vector Laboratories, Burlingame, CA, U.S.A.), both diluted at 1:200 in PBS-T. Cultures were then washed in PBS, and staining was completed using an avidin-biotin-peroxidase complex (ABC; Vectastain Elite ABC kits; Vector Laboratories). After rinsing in PBS, cultures were incubated for 5-10 min in PBS containing 0.05% 3,3′ -diaminobenzidine and 0.002% H2O2. Then, the coverslips were extensively rinsed, dehydrated, and mounted on glass slides with Eukitt. Cultures treated identically, except for replacing the primary antibody by nonimmune serum from the adequate animal, served as control for antibody specificity. This treatment yielded nondetectable staining.

Microscopic examination for quantification was performed under bright-field illumination using a quadratic counting grid placed in the ocular of the microscope (Olympus model BH-2). Counts of labeled cells were made using a 25× objective lens in a total population of at least 3,000 cells. The percentage of immunoreactive neurons was determined by counting the number of labeled cells and by reporting this value relative to the total number of cells. Data represent at least two independent experiments, each comprising triplicate culture preparations. Results are expressed as percentages of control. Statistical analysis was performed using Student's t test.

Double-labeling experiments. For colocalization studies of GABA with calretinin or calbindin, cells on glass coverslips were fixed in a mixture of 4% paraformaldehyde and 0.1% glutaraldehyde at room temperature for 25 min. After washing in PBS-T, coverslips were incubated in 4% normal donkey serum diluted in PBS-T for 50 min. This step was followed by an overnight incubation at room temperature in a cocktail of primary antibodies, diluted in PBS-T as follows: cocktail 1, rabbit anti-GABA (Sigma; 1:5,000) and mouse monoclonal anti-calbindin (Swant; 1:1,000); cocktail 2, mouse monoclonal anti-GABA (Chemicon; 1:1,000) and rabbit anti-calretinin (Swant; 1:5,000); and cocktail 3, rabbit anti-calretinin (Swant; 1:5,000) and mouse monoclonal anti-calbindin (Swant; 1:1,000). The next day, after two 7-min rinses in PBS-T and one 7-min rinse in PBS, coverslips were incubated in the following mixtures of secondary antibodies for 20 min: mixture 1, fluorescein isothiocyanate-conjugated anti-mouse IgG and CY3-conjugated anti-rabbit IgG (1:1,000 final concentration of each); and mixture 2, fluorescein isothiocyanate-conjugated anti-rabbit IgG and CY3-conjugated anti-mouse IgG (1:1,000). All secondary antibodies were raised in donkeys (Jackson ImmunoResearch Laboratories, West Grove, PA, U.S.A.). Eventually, coverslips were washed and mounted on glass slides with Vectashield (Vector Laboratories).

Stained cells were visualized under a Leitz (Wetzlar, Germany) fluorescence microscope. The quantitative analysis of double-labeled neurons was performed by checking at least 100 cells on each coverslip. Experiments were performed in triplicates.

Effects of BDNF on number of calbindin- and calretinin-immunoreactive neurons

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. RESULTS
  5. Effects of BDNF on number of calbindin- and calretinin-immunoreactive neurons
  6. Effects of BDNF on levels of calbindin and calretinin mRNAs
  7. DISCUSSION
  8. Acknowledgements

Regulation of calbindin and calretinin expression by BDNF was examined in primary cultures of mouse cortical neurons using immunocytochemical staining and northern blot analysis. In control cultures, the percentage of calbindin- and calretinin-positive neurons was 5.85 ± 0.4 (n = 16) and 6.94 ± 0.25% (n = 18) of the total cell population, respectively. Treatment of cultured cortical neurons with increasing concentrations of BDNF resulted in a concentration-dependent increase in the number of calbindin-positive neurons compared with control cultures (Fig. 1). For instance, the number of calbindin-containing neurons almost doubled after stimulation with 5 ng/ml BDNF reaching 10.88 ± 0.71% (n = 8) of the total neuronal population.

image

Figure 1. BDNF increases the number of calbindin-immunoreactive neurons. Cultures of cortical neurons were treated for 5 days in the presence of increasing concentrations of BDNF. A: Immunocytochemical detection of calbindin-positive neurons in control (Ctrl) cultures and in cultures treated with 5 ng/ml BDNF. Bar = 20 μm. B: Quantitative analysis of calbindin-immunoreactive neurons was performed as described in Experimental Procedures. Data are mean ± SEM (bars) percentages of Ctrl values from three to eight determinations from three independent experiments. In Ctrl cultures, the percentage of calbindin-positive neurons was found to represent 5.85 ± 0.4% (n = 16) of the total cell population. *p < 0.05, ***p < 0.001 by Student's t test between Ctrl and BDNF-treated cultures.

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In contrast to its effect on calbindin expression, BDNF induced a marked decrease in the number of calretinin-positive neurons (Fig. 2). For instance, daily treatment with 5 ng/ml BDNF for 5 days reduced by 60% the number of cortical neurons expressing calretinin as the number of calretinin-positive cells dropped to 2.68 ± 0.31% (n = 12).

image

Figure 2. BDNF decreases the number of calretinin-immunoreactive neurons. Cultures of cortical neurons were treated for 5 days in the presence of increasing concentrations of BDNF. A: Immunocytochemical detection of calretinin-positive neurons in control (Ctrl) cultures and in cultures treated with 5 ng/ml BDNF. Bar = 20 μm. B: Quantitative analysis of calretinin-immunoreactive neurons was performed as described in Experimental Procedures. Data are mean ± SEM (bars) percentages of Ctrl values from six to 18 determinations from four independent experiments. In Ctrl cultures, the percentage of calretinin-positive neurons was found to represent 6.94 ± 0.25% (n = 18) of the total cell population. ***p < 0.001 by Student's t test between Ctrl and BDNF-treated cultures.

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Parvalbumin-positive neurons were detected neither in control nor in BDNF-treated cultures.

Considering the striking opposite effects of BDNF on the expression of these two cytosolic calcium-binding proteins, it was of importance to determine whether or not these proteins were localized in the same neuronal population. Double-labeling experiments were carried out to investigate the degree of colocalization of calbindin and calretinin. In agreement with previous studies (Celio, 1990; Resibois and Rogers, 1992; Gonchar and Burkhalter, 1997), our results revealed that only 6.2 ± 0.9% (n = 4) of calbindin-containing neurons coexpressed calretinin. This percentage of colocalization was not altered by treatment with BDNF (data not shown), indicating that the opposite regulation of these two calcium-binding proteins does not occur within the same neurons.

Although calbindin and calretinin are distributed in mainly nonoverlapping neuronal populations, they are known to be colocalized, at least in part, with the inhibitory neurotransmitter GABA (Rogers, 1992; Kubota et al., 1994; Gonchar and Burkhalter, 1997; Miettinen et al., 1997). We therefore examined their degree of colocalization with GABA with particular emphasis on the effects exerted by BDNF. Double-labeling analysis revealed that calbindin was highly colocalized with GABA because 92.1 ± 3.6% (n = 4) of calbindin-positive neurons expressed GABA, whereas only 37.8 ± 1.5% (n = 5) of calretinin-positive neurons were GABA-immunoreactive. After treatment with 5 ng/ml BDNF, the percentage of colocalization of calbindin with GABA was not changed (94.8 ± 1.1%; n = 4), whereas the percentage of colocalization of GABA with calretinin was significantly increased from 37.8 ± 1.5 to 50.1 ± 1.6% (n = 5, p < 0.001).

Calbindin up-regulation by BDNF was not associated with an increase in the number of GABAergic neurons. Indeed, the percentage of GABAergic neurons (11.3 ± 0.6%, n = 6) remained unchanged after exposure to 5 ng/ml BDNF (11.8 ± 0.5%, n = 6), which is consistent with the unaffected expression of GABA in the cerebral cortex and hippocampus of BDNF knockout mice (Jones et al., 1994).

Effects of BDNF on levels of calbindin and calretinin mRNAs

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. RESULTS
  5. Effects of BDNF on number of calbindin- and calretinin-immunoreactive neurons
  6. Effects of BDNF on levels of calbindin and calretinin mRNAs
  7. DISCUSSION
  8. Acknowledgements

Because BDNF has been reported to increase intracellular Ca2+ levels (Berninger et al., 1993; Marsh and Palfrey, 1996) and that antibody recognition of calbindin and calretinin depends on their calcium-binding status (Winsky and Kuznicki, 1996), we examined, by northern blot experiments, the levels of mRNAs encoding these two calcium-binding proteins. Consistent with immunocytochemical analysis, BDNF elicited a concentration-dependent increase in the levels of calbindin mRNA (Fig. 3). For instance, levels of calbindin mRNA increased by about fourfold after treatment with 5 ng/ml BDNF. In marked contrast to calbindin mRNA expression, levels of calretinin mRNA were reduced by BDNF (Fig. 4). As evidenced in Fig. 4, calretinin mRNA levels were decreased by 85.6 ± 2.7% (n = 3) after exposure to 5 ng/ml BDNF. Confirming our immunocytochemical results, northern blot analysis did not reveal parvalbumin mRNA expression in control or in BDNF-treated cultures (data not shown).

image

Figure 3. BDNF stimulates calbindin mRNA expression in cultured cortical neurons. A: Cultures of cortical neurons were treated for 5 days in the presence of increasing concentrations of BDNF. Extraction of total RNA and northern blot analysis were performed as described in Experimental Procedures. In each lane 10 μg of total RNA was loaded. Ctrl, control. B: Quantitative analysis of calbindin mRNA levels after stimulation with increasing concentrations of BDNF. Quantitative analysis was performed by densitometric scanning as described in Experimental Procedures. Data are mean ± SEM (bars) percentages of Ctrl values from three independent experiments.

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image

Figure 4. BDNF inhibits calretinin mRNA expression in cultured cortical neurons. A: Cultures of cortical neurons were treated for 5 days in the presence of increasing concentrations of BDNF. Extraction of total RNA and northern blot analysis were performed as described in Experimental Procedures. In each lane 10 μg of total RNA was loaded. Ctrl, control. B: Quantitative analysis of calretinin mRNA levels after stimulation with increasing concentrations of BDNF. Quantitative analysis was performed by densitometric scanning as described in Experimental Procedures. Data are mean ± SEM (bars) percentages of Ctrl values from three independent experiments.

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Altogether, these results strongly suggest that alterations in the number of calbindin- and calretinin-containing neurons reflect changes in the levels of calbindin and calretinin mRNAs.

Effects of other neurotrophins on expression of calbindin and calretinin

To characterize the effects of other members of the neurotrophin family on the differential regulation of calbindin and calretinin expression, we tested the effects of NGF, NT-3, and NT-4. As illustrated in Figs. 5 and 6, NT-4, which also acts by activating TrkB receptors (Klein et al., 1992), exerted effects comparable to those of BDNF. Indeed, NT-4 decreased calretinin mRNA levels and the number of calretinin-expressing neurons (Fig. 5), whereas it increased the expression of calbindin mRNA and the number of calbindin-immunoreactive neurons (Fig. 6). In contrast, NGF did not regulate the expression of these two calcium-binding proteins (Figs. 5 and 6). Similarly to BDNF and NT-4, treatment of cultured cortical neurons with NT-3 also led to a reduction in calretinin mRNA expression and in the number of calretinin-positive neurons (Fig. 5). However, unlike BDNF and NT-4, NT-3 did not increase calbindin mRNA expression or the number of calbindin-immunoreactive neurons (Fig. 6).

image

Figure 5. Effects of neurotrophins on number of calretinin-immunoreactive neurons and on level of calretinin mRNA. Cultures of cortical neurons were treated for 5 days in the presence of NGF, BDNF, NT-3, or NT-4 added at a concentration of 5 ng/ml. A: Immunocytochemical detection of calretinin-immunoreactive neurons and cell counting were performed as described in Experimental Procedures. Data are mean ± SEM (bars) percentages of control (Ctrl) values from six determinations from two independent experiments. In Ctrl cultures, the percentage of calretinin-positive neurons was found to represent 7.52 ± 0.92% (n = 6) of the total cell population. **p < 0.01 by Student's t test between Ctrl and treated cultures. B: Northern blot analysis of calretinin mRNA expression after treatment with NGF, BDNF, NT-3, or NT-4. Extraction of total RNA and northern blot analysis were performed as described in Experimental Procedures. In each lane 10 μg of total RNA was loaded. C: Quantitative analysis of calretinin mRNA levels after stimulation with NGF, BDNF, NT-3, or NT-4. Quantitative analysis was performed by densitometric scanning as described in Experimental Procedures. Data are mean ± SEM (bars) percentages of control from three independent experiments.

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image

Figure 6. Effects of neurotrophins on number of calbindin-immunoreactive neurons and on level of calbindin mRNA. Cultures of cortical neurons were treated for 5 days in the presence of NGF, BDNF, NT-3, or NT-4 added at a concentration of 5 ng/ml. A: Immunocytochemical detection of calbindin-immunoreactive neurons and cell counting were performed as described in Experimental Procedures. Data are mean ± SEM (bars) percentages of control (Ctrl) values from six determinations from two independent experiments. In Ctrl cultures the percentage of calbindin-positive neurons was found to represent 6.49 ± 0.72% (n = 6) of the total cell population. **p < 0.01 by Student's t test between Ctrl and treated cultures. B: Northern blot analysis of calbindin mRNA expression after treatment with NGF, BDNF, NT-3, or NT-4. Extraction of total RNA and northern blot analysis were performed as described in Experimental Procedures. In each lane 10 μg of total RNA was loaded. C: Quantitative analysis of calbindin mRNA levels after stimulation with NGF, BDNF, NT-3, or NT-4. Quantitative analysis was performed by densitometric scanning as described in Experimental Procedures. Data are mean ± SEM (bars) percentages of Ctrl values from three independent experiments.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. RESULTS
  5. Effects of BDNF on number of calbindin- and calretinin-immunoreactive neurons
  6. Effects of BDNF on levels of calbindin and calretinin mRNAs
  7. DISCUSSION
  8. Acknowledgements

The purpose of the present study was to investigate the regulation of the expression of three calcium-binding proteins, i.e., calretinin, calbindin, and parvalbumin, by BDNF and by other members of the neurotrophin family.

The main result reported here is an opposite regulation by BDNF and NT-4 of calretinin and calbindin expression in cultured cortical neurons. Indeed, in response to chronic treatment by BDNF or NT-4, the expression of calbindin mRNA and the number of calbindin-immunoreactive neurons are strongly up-regulated (Figs. 1, 3, and 6), whereas the expression of calretinin mRNA and the number of calretinin-immunoreactive neurons are remarkably decreased (Figs. 2, 4, and 5). Similar treatment of cortical neurons with NT-3 also leads to a marked reduction in the levels of calretinin mRNA and in the number of calretinin-positive neurons (Fig. 5). However, in contrast to BDNF and NT-4, NT-3 does not increase calbindin expression (Fig. 6). The lack of parvalbumin, the third calcium-binding protein investigated in this study, may be ascribed to the delayed appearance of parvalbumin as compared with calretinin and calbindin in rodent cerebral cortex in vivo. Indeed, the latter appear in the rat cortical anlage at embryonic day 14 (Enderlin et al., 1987; Sanchez et al., 1992; Fonseca et al., 1995), whereas parvalbumin is first detected after postnatal week 1 (Solbach and Celio, 1991; Sanchez et al., 1992).

Neurotrophins regulate cell functions by binding to and activating specific Trks. NGF is the specific ligand for TrkA, whereas BDNF and NT-4 preferentially activate TrkB (Bothwell, 1995). NT-3 binds to TrkC and to a lesser extent to TrkB (Soppet et al., 1991). TrkB and TrkC receptors have been reported to be abundant during mouse brain embryonic and postnatal development (Klein et al., 1990; Escandon et al., 1994; Lamballe et al., 1994), whereas levels of TrkA were found to be low (Yan and Johnson, 1988; Koh and Loy, 1989; Martin-Zanca et al., 1990). Consistent with these in vivo studies, northern blot analysis of Trk receptors in our 6 days in vitro cultures has revealed the presence of TrkB and TrkC but not TrkA mRNAs (data not shown), providing an explanation for the lack of response of this preparation to NGF (Figs. 5 and 6). The similarity between the effects of BDNF and NT-4 on the regulation of calbindin and calretinin expression is consistent with their actions via TrkB receptors. As to the absence of calbindin regulation by NT-3, it may be ascribed to the fact that in our cultures calbindin is largely confined to GABAergic neurons (92.1 ± 3.5%, n = 4), which do not colocalize TrkC mRNA, as shown in the rat visual cortex (Gorba and Wahle, 1999).

There is a general agreement that many calbindin- and calretinin-containing neurons coexpress GABA in rat cerebral cortex. Although the colocalization of calretinin with GABA differs between studies (Rogers, 1992; Gonchar and Burkhalter, 1997; Miettinen et al., 1997), fewer discrepancies exist in the degree of overlap between calbindin and GABA, which have been repeatedly observed to be highly colocalized (Rogers, 1992; Kubota et al., 1994; Gonchar and Burkhalter, 1997). Our double-labeling experiments reveal a high percentage of cells coexpressing calbindin and GABA (92.1 ± 3.5%, n = 4). The still important coexpression of GABA with calbindin after treatment with BDNF (94.8 ± 1.1%, n = 4) indicates that up-regulation of calbindin expression by BDNF (Figs. 1, 3, and 6) takes place in GABAergic neurons. Thus, after exposure to BDNF, the vast majority of GABAergic neurons (∼90%) also contain calbindin. In marked contrast to calbindin, the ratio of calretinin-positive neurons coexpressing GABA is only 37.8 ± 1.5% (n = 4), suggesting that the majority of calretinin-labeled cells correspond to non-GABAergic neurons. In agreement with these observations, calretinin has been shown to be transiently expressed in certain GABA-negative populations during cortical (Fonseca et al., 1995; Yan et al., 1995) and hippocampal (Jiang and Swann, 1997) ontogenesis. Although TrkB receptors are expressed on both pyramidal and GABAergic cells (Cabelli et al., 1996; Cellerino et al., 1996; Gorba and Wahle, 1999), the significant increase in the colocalization of GABA with calretinin after treatment with BDNF (50.1 ± 1.6%; n = 6) indicates that down-regulation of calretinin expression by BDNF (Figs. 2, 4, and 5) preferentially occurs in non-GABAergic neurons. Thus, according to the neurochemical phenotype of the target cell, TrkB receptor activation by BDNF or NT-4 leads to an opposite regulation of calbindin and calretinin via distinct transduction pathways.

Although calbindin has been previously shown to be up-regulated by BDNF in vivo and in vitro (Ip et al., 1993; Jones et al., 1994; Widmer and Hefti, 1994), this is the first time that inhibition of calretinin expression by BDNF has been observed. This down-regulation of calretinin expression by BDNF may have a physiological relevance during development. Indeed, it has been reported that, during the second and third postnatal week, the expression of calretinin decreases sharply to reach adult levels in the rat cerebral cortex (Schierle et al., 1997). It is interesting that studies in the rat visual cortex have shown that the expression of BDNF mRNA is markedly increased during the same period (Castren et al., 1992; Bozzi et al., 1995; Schoups et al., 1995). Similar decreases in calretinin levels have been reported in the rat hippocampus after the second postnatal week (Villa et al., 1994; Jiang and Swann, 1997), where BDNF expression has been shown to reach high levels relative to earlier stages of development (Maisonpierre et al., 1990; Friedman et al., 1991). The temporal correlation between the appearance of BDNF and the disappearance of calretinin, together with our observations, strongly suggests that the ontogenetic down-regulation of calretinin observed in the cerebral cortex in vivo may result from a BDNF-mediated mechanism. However, a definite link between BDNF and calretinin down-regulation in vivo awaits further experiments using BDNF knockout mice.

Acknowledgements

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. RESULTS
  5. Effects of BDNF on number of calbindin- and calretinin-immunoreactive neurons
  6. Effects of BDNF on levels of calbindin and calretinin mRNAs
  7. DISCUSSION
  8. Acknowledgements

The authors wish to thank Igor Allaman for expert advice on northern blot analysis. This work was supported by the Swiss National Science Foundation with grants 3100-050806.97 to J.-L.M. and 3100-40565.94 to P.J.M.

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