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First published online October 22, 2003
doi: 10.1242/10.1242/dev.00826

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Accelerated dendritic development of rat cortical pyramidal cells and interneurons after biolistic transfection with BDNF and NT4/5

AG Entwicklungsneurobiologie, Fakultät für Biologie, Ruhr-Universität, ND 6/56a, D-44780 Bochum, Germany

View larger version (36K): [in a new window]   Fig. 7. Quantification of dendritic parameters of adult pyramidal neurons of layers II/III, V and VI from active (white bars) and activity-deprived (hatched bars) OTC. Neither length nor segments numbers of apical and basal dendrites differed significantly. By contrast, spine density was significantly increased on apical and basal dendrites of pyramidal cells from layers II/III and VI. **=P<0.01; ***=P<0.001. The numbers in the bars represent the number of dendrites analyzed. For apical dendrites, they equal the number of neurons analyzed.

View larger version (27K): [in a new window]   Fig. 1. Representative pyramidal neurons from layers II/III, V and VI transfected with EGFP, BDNF/EGFP and NT4/5/EGFP. (A) Neurons of the early time window, DIV 0-5. Only layer VI neurons displayed more profuse dendritic trees. (B) Neurons of the later time window, DIV 5-10. Now BDNF and NT4/5 strongly accelerated the development of apical and basal dendrites. Note that neurotrophin transfectants at 5 DIV appear quite similar to EGFP control transfectants at 10 DIV.

View larger version (29K): [in a new window]   Fig. 2. The early time window: quantification of dendritic length and number of segments for apical and basal dendrites of pyramidal neurons in layers II/III, V and VI after overexpression of EGFP, BDNF/EGFP and NT4/5/EGFP. *=P<0.05; **=P<0.01, Mann-Whitney-U-tests versus EGFP control. The numbers in the bars represent the number of dendrites analyzed. For apical dendrites they equal the number of neurons analyzed.

View larger version (34K): [in a new window]   Fig. 3. The later time window: quantification of dendritic length and number of segments for apical and basal dendrites of pyramidal neurons in layers II/III, V and VI after overexpression of EGFP, BDNF/EGFP and NT4/5/EGFP. *=P<0.05; **=P<0.01, Mann-Whitney-U-tests versus EGFP control. The numbers in the bars represent the number of dendrites analyzed. For apical dendrites, they equal the number of neurons analyzed. See also Table 1 for total basal dendritic length and segment numbers.

View larger version (153K): [in a new window]   Fig. 6. Representative pyramidal cells of layers II/III from adult OTC. (A-C) Neuron and high magnifications of spiny dendrites from active OTC. (D-F) Neuron and high magnifications of spiny dendrites from activity-deprived OTC. Scale bar: 150 µm.

View larger version (50K): [in a new window]   Fig. 4. Photomicrographs of representative multipolar interneurons after transfection with (A) EGFP, (B) BDNF/EGFP and (C) NT4/5/EGFP. Scale bar: 100 µm.

View larger version (25K): [in a new window]   Fig. 5. Quantification of dendritic parameters of multipolar interneurons from layers II/III, V and VI after overexpression of EGFP, BDNF/EGFP and NT4/5/EGFP during the later time window, DIV 5-10. For direct comparison, dendritic parameters of biocytin-filled multipolar interneurons from adult spontaneously active OTC were included. The neurotrophin transfectants at 10 DIV displayed number of segments and maximum branch orders close to adult interneurons, whereas dendritic length was still far from being mature. *=P<0.05; **=P<0.01; ***=P<0.001. The numbers in the bars represent the number of dendrites analyzed.

View larger version (37K): [in a new window]   Fig. 8. NT4/5 but not BDNF is essential for dendritogenesis. Apical and basal dendritic length and number of segments were quantified for pyramidal neurons in layer VI in the following conditions (indicated on the abscissa): SBA+, EGFP transfectants in active OTC; SBA–, EGFP transfectants in deprived OTC; BDNF SBA–, BDNF transfectants in deprived OTC; NT4 SBA–, NT4/5 transfectants in deprived OTC; anti-BDNF SBA+, EGFP transfectants in active OTC treated with neutralizing antibodies against BDNF from 5-10 DIV; antiNT4 SBA+, EGFP transfectants in active OTC treated with neutralizing antibodies against NT4/5 from 5-10 DIV; K252a, EGFP transfectants in active OTC treated with K252a from 5-10 DIV; BDNF K252a, BDNF transfectants in active OTC treated with K252a from 5-10 DIV; NT4 K252a, NT4/5 transfectants in active OTC treated with K252a from 5-10 DIV. The numbers in the bars represent the number of dendrites analyzed. For apical dendrites they equal the number of neurons analyzed. *=P<0.05; **=P<0.01, Mann-Whitney-U-tests versus SBA+ condition.

View larger version (16K): [in a new window]   Fig. 9. Developmental mRNA expression of BDNF, NT3 and NT4/5 in active (SBA+, black bars) and activity-deprived (SBA–, white bars) OTC. (A) BDNF mRNA expression increased with age in active OTC, but remained low in deprived OTC. (B) NT3 mRNA expression was slightly upregulated in young deprived OTC and decreased until 45 DIV. (C) NT4/5 mRNA expression was strongly upregulated in 10 and 20 DIV activity-deprived OTC. The expression levels of the three mRNAs in the 10 DIV SBA+ condition were set to 1.

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