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First published online 11 July 2006
doi: 10.1242/jcs.03042

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Proliferation of cerebellar precursor cells is negatively regulated by nitric oxide in newborn rat

¹ Department of Human and General Physiology, University of Bologna, Piazza di Porta San Donato 2, 40126 Bologna, Italy
² Department of Biology, University of Bologna, via Selmi 3, 40126 Bologna, Italy

View larger version (19K): [in a new window]   Fig. 1. Activity and expression of nNOS in cerebellar development of control and L-NAME-treated rat pups. Pups were s.c. injected with L-NAME (60 mg/kg/day, divided in two daily administrations) for various neonatal intervals (P1-P3, P3-P5, P5-P7) while control pups received vehicle. Animals were killed on the last day of treatment. (A) Maturation of calcium-dependent NOS activity in developing cerebella (white bars) and almost complete inhibition obtained by L-NAME treatment (black bars). Values are the mean ± s.e.m. of at least three experiments for each time point. *P<0.05, compared with the control at P3, #P<0.001 compared with the corresponding control (Bonferroni's test after ANOVA). (B) Total RNA was extracted from cerebella of control and L-NAME-treated rat pups and equal amounts of RNA were used for real-time PCR performed with specific primers for nNOS RNA as described in the Materials and Methods section. These results, expressed as a percentage of control at P3 are the mean ± s.e.m. of four different experiments performed in triplicate. *P<0.05, **P<0.01 compared to P3 (Bonferroni's test after ANOVA). (C) In western blot experiments, cerebellar extracts from control and L-NAME-treated rat pups were probed with an antibody specific for nNOS. Bands were quantified by optical densitometry and normalized for the amount of ß-actin. These results are the mean ± s.e.m. of four different experiments. *P<0.05, **P<0.01 compared with P3 (Bonferroni's test after ANOVA).

View larger version (22K): [in a new window]   Fig. 2. Expression of soluble guanylyl cyclase (sGC) subunits (1, 2 and ß1) during cerebellar development. Total RNA was extracted from cerebella (A) or from freshly isolated cerebellar granule cells (FIC) (B) of rat pups and equal amounts of RNA were used for real-time PCR performed with specific primers for guanylyl cyclase subunits as described in Materials and Methods section. The results, expressed as % of control at P3, are the mean ± s.e.m. of four different experiments performed in triplicate. *P<0.01, **P<0.001 compared to P3 (Bonferroni's test after ANOVA). (C) Concentration of cerebellar cGMP determined by an enzyme-linked immunoassay. Pups were treated with L-NAME (60 mg/kg/day) for various neonatal intervals (P1-P3, P3-P5, P5-P7) or with the guanylyl cyclase inhibitor ODQ (5 mg/kg/day) in two daily administrations for various neonatal intervals (P1-P3, P3-P5, P5-P7), while control pups received vehicle. Values are the mean ± s.e.m. of three different experiments *P<0.01 compared with P3, #P<0.01, ##P<0.001 compared with the corresponding control (Bonferroni's test after ANOVA).

View larger version (57K): [in a new window]   Fig. 3. Effect of blockade of NO production on cell proliferation. (A) Cell proliferation was measured on cerebellar homogenates from rat pups treated with L-NAME (60 mg/kg/day) or with ODQ (5 mg/kg/day) for various neonatal intervals (P1-P2, P1-P3, P3-P5, P5-P7), while control pups received vehicle. Two hours prior to sacrifice, pups were s.c. injected with [3H]thymidine (5 µCi/g body weight). The % of [3H]thymidine incorporation into DNA was expressed as dpm/µg protein. Values are the mean ± s.e.m. of four different experiments *P<0.05, **P<0.01 compared with the corresponding controls of the same age (dotted line) (Bonferroni's test after ANOVA). (B) To assess apoptosis in the developing cerebellum, cerebellar homogenates from pups of various postnatal ages (3, 5, 7 and 11 days) were monitored, using a sandwich ELISA method, for histone-associated DNA fragments in the cytoplasm. L-NAME-treated pups received the usual drug dosage at the following intervals: P1-P3, P3-P5, P5-P7, P9-P11. Values are the mean ± s.e.m. of three independent experiments (Bonferroni's test after ANOVA). (C,D) BrdU-labelled cells in the cerebellum of 3-day-old pups after BrdU administration at P3, in a control rat (C) and a rat treated with L-NAME from P1 to P3 (D). Bar, 100 µm. (E) Density of BrdU-labelled cells (number of cells/mm2) in the external granular layer (EGL) and internal granular layer (IGL) of the cerebellum of control and L-NAME P1-P3 treated 3-day-old rat pups. Values are the mean ± s.e.m. of three experiments. *P<0.01, compared with the control (Student's t-test).

View larger version (73K): [in a new window]   Fig. 4. Survival of the postnatally generated cells. (A,B) BrdU-labelled cells in the molecular layer (ML) or the granular layer (GL) of the cerebellum of 60-day-old rats after BrdU administration at P3, in a control rat (A) and a rat treated with L-NAME from P1 to P3 (B). Bar, 200 µm. (C) Density of BrdU-labelled cells (number of cells/mm2) in the molecular layer (ML) and granular layer (GL) of the cerebellum of control rats and rats treated with L-NAME from P1 to P3 rats. (D) Density of labelled cells in 60-day-old animals treated with L-NAME or saline from P5 to P7 and injected with BrdU at P7. For C and D, bars are the mean ± s.e.m. of three experiments. *P<0.01, compared to control (Student's t-test).

View larger version (134K): [in a new window]   Fig. 5. Phenotype of neonatally BrdU-labelled cells in 60-day-old rats. Merged confocal microscope images of immunoreactivity for BrdU (in green), and NeuN (in red) (A,B) and for BrdU (in green), and GFAP (in red) (C) in 40 µm thick sections of cerebellum of 60-day-old animals; colocalization appears yellow. (A) Control, (B,C) P1-P3 L-NAME-treated rats. These animals received on P3 a BrdU injection (100 µg/g body weight) after 3 days administration of L-NAME or vehicle and were sacrificed at 60 days of age. (D) Number of BrdU and NeuN (white) or BrdU and GFAP (hatched) labelled cells expressed as a percentage of total number of BrdU-labelled cells. The black part of the bars represents the percentage of cells that were of indeterminate phenotype. Values are the mean ± s.e.m. of three experiments.

View larger version (56K): [in a new window]   Fig. 6. Expression of Myc (A) and cyclin D1 (B) in cerebellar homogenates of rat pups treated with L-NAME (60 mg/kg/day) for various neonatal intervals (P1-P3, P5-P7) and control pups. Total RNA was extracted from cerebella and equal amounts of RNA were used for real-time PCR performed with specific primers for Myc and cyclin D1, as described in the Materials and Methods section. These results, expressed as percentage of control at P3, are the mean ± s.e.m. of three different experiments performed in triplicate. *P<0.01, **P<0.001 compared with control, #P<0.01 compared with P3 control (Bonferroni's test after ANOVA). (C) Total cerebellar lysates from L-NAME-treated (P1-P3 and P5-P7) and control rat pups were immunoblotted with an antibody to Myc and quantification of bands was obtained with respect to ß-actin content. Values are the mean ± s.e.m. of three experiments. **P<0.001 compared to control, #P<0.01 compared to P3 control (Bonferroni's test after ANOVA). (D) Nuclear and cytoplasmic cerebellar lysates from L-NAME treated (P1-P3) and control rat pups were separately immunoblotted with an antibody to Myc and quantified respect to ß-actin content. Bars are the mean ± s.e.m. of three experiments. *P<0.05, **P<0.001 compared to cytoplasmic extracts, (Bonferroni's test after ANOVA). (E,F) Expression of Myc protein in the cerebellum of control (E) and P1-P3 L-NAME-treated (F) rat pups. Immunohistochemistry with an anti-Myc antibody reveals an increased stain in the external granular layer of L-NAME-treated pup. Bar, 40 µm.

View larger version (56K): [in a new window]   Fig. 7. (A) Freshly plated CGC cultures prepared from P2 cerebella were stimulated with L-NAME (1 mM), ODQ (5 µM) or KT5823 (500 nM), for 20 hours. BrdU (10 µM) was added for the last 6 hours, after which time cells were processed for BrdU incorporation by an ELISA method. Values are the mean ± s.e.m. of three experiments. *P<0.05, compared to control (Bonferroni's test after ANOVA). (B,C) Colour merged images of double immunofluorescence for III beta-tubulin (green) and BrdU (red) of control (B) and L-NAME-treated (C) CGC cultures. Plated CGC cultures were stimulated with L-NAME (1 mM) for 20 hours. BrdU (10 µM) was added for the last 6 hours, after which time cells were processed for immunofluorescence. Arrowheads point to III beta-tubulin-BrdU labelled cells. Bar, 30 µm. (D) Percentage of BrdU-labelled cells positive for the early neuronal marker, III beta-tubulin, or the glial marker, GFAP in the same cultures as A-C. Counting was done in random fields of control and L-NAME-treated cultures from two independent experiments. *P<0.01, compared to control (Bonferroni's test after ANOVA). (E) BrdU incorporation during the last 6 hours of culture was measured in CGC by ELISA method after 20 hours in culture in the presence or absence of L-NAME (1 mM) and/or Shh (3 µg/ml). Data are expressed as the mean ± s.e.m. of three experiments. *P<0.05, **P<0.01, compared to control (Bonferroni's test after ANOVA). (F) Expression of Myc in CGC cultures treated with L-NAME (1 mM) and/or Shh (3 µg/ml) for 12 hours. Total RNA was extracted from CGC cultures and equal amounts were used for real-time PCR reaction performed with specific primers for Myc, as described in the Materials and Methods section. The results, expressed as percentage of control, are the mean ± s.e.m. of two different experiments performed in triplicate. *P<0.05, **P<0.01 (Bonferroni's test after ANOVA).

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