Neurotrophic activity of pituitary adenylate cyclase-activating polypeptide on rat cerebellar cortex during development - PubMed (original) (raw)

Neurotrophic activity of pituitary adenylate cyclase-activating polypeptide on rat cerebellar cortex during development

D Vaudry et al. Proc Natl Acad Sci U S A. 1999.

Abstract

High concentrations of pituitary adenylate cyclase-activating polypeptide (PACAP) receptors are present in the external granule cell layer of the rat cerebellum during postnatal development. In vitro studies have shown that PACAP promotes cell survival and neurite outgrowth on immature cerebellar granule cells in primary culture. In the present study, we have investigated the effect of PACAP on the development of the cerebellar cortex of 8-day-old rats. Incubation of cultured granule cells for 12 or 18 h with PACAP provoked a significant increase in the rate of incorporation of [(3)H]thymidine in cultured granule cells, suggesting that PACAP could stimulate the proliferation of granule cells. After 96 h of treatment, in vivo administration of PACAP provoked a transient increase in the number of granule cells in the molecular layer and in the internal granule cell layer. In contrast, PACAP did not affect the number of Purkinje cells. The augmentation of the number of granule cells evoked by PACAP was significantly inhibited by the PACAP receptor antagonist PACAP(6-38). Administration of PACAP also caused a significant increase in the volume of the cerebellar cortex. The present study provides evidence that PACAP can act in vivo as a trophic factor during rat brain development. Our data indicate that PACAP increases proliferation and/or inhibits programmed cell death of granule cells, as well as stimulating neuronal migration from the external granule cell layer toward the internal granule cell layer.

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Figures

Figure 1

Figure 1

Effect of PACAP on [3H]thymidine incorporation by cultured cerebellar granule cells. (A and B) Effect of graded concentrations of PACAP on [3H]thymidine incorporation. Cerebellar granule cells were exposed to PACAP for 6 h (A) or 18 h (B). Data are expressed as percentage of control. (C) Time-course of the effect of PACAP on [3H]thymidine incorporation. Cerebellar granule cells were incubated in control conditions (○) or in the presence of 10−9 M PACAP (●) for durations ranging from 6 to 24 h. Each value represents the mean ± SEM of at least three independent experiments performed in triplicate. ∗, P < 0.05 vs. control; ∗∗, P < 0.01 vs. control; ∗∗∗, P < 0.001 vs. control; #, P < 0.05 vs. time 6 h; ##, P < 0.01 vs. time 6 h.

Figure 2

Figure 2

Effects of graded doses of PACAP on the histogenesis of the Sim in P12 rat cerebella. (A) Typical Cresyl Violet staining of a rat cerebellum slice at the caudal extremity of the colliculi and the rostral extremity of lobule 1, showing the type of section used for measurements with the confocal laser-scanning microscope. (Bar = 1.4 mm.) (B) Higher magnification of a Sim showing the series of fields that were used for the measurement of the thickness of the EGL, the molecular layer (Mol), and the IGL, as well as for the measurement of the density of granule cells in the molecular layer and IGL. (Bar = 400 μm.) (C) Quantification of the thickness of the EGL, molecular layer, and IGL from the Sim of P12 rats. (Bar = 100 μm.) Each value represents the mean ± SEM of a representative experiment performed in triplicate. ∗, P < 0.05 vs. control; ∗∗, P < 0.01 vs. control.

Figure 3

Figure 3

Time-course of the effect of PACAP on the thickness of the EGL (A), the thickness of the molecular layer (B), the thickness of the IGL (C), the number of granule cells crossing the molecular layer (D), and the number of granule cells in the IGL (E). P8 rats were treated with saline (open bars), 0.01 μg of PACAP (hatched bars), or 1 μg of PACAP (black bars) up to P16. Each value represents the mean ± SEM of at least three experiments performed in triplicate. ∗, P < 0.05 vs. control; ∗∗, P < 0.01 vs. control.

Figure 4

Figure 4

Effects of the PACAP antagonist PACAP(6–38) on the PACAP-induced increase of the thickness (A) and number (B) of cells in the IGL. P8 rats were treated with saline, 0.01 μg of PACAP, and/or 1 μg of PACAP(6–38). Injections were renewed at P10, and animals were killed at P12. Data are expressed as percentage of control. Each value represents the mean ± SEM of three experiments performed in triplicate. ∗∗, P < 0.01; ∗∗∗, P < 0.001.

Figure 5

Figure 5

Time-course of the effect of PACAP on the volume of the cerebellar medulla (A), the cerebellar cortex (B), and the whole cerebellum (C). P8 rats were treated with saline (open bars), 0.01 μg of PACAP (hatched bars), or 1 μg of PACAP (black bars) up to P16. Each value represents the mean ± SEM of at least three experiments performed in triplicate. ∗, P < 0.05 vs. control.

Figure 6

Figure 6

Displacement of [125I]PACAP27 binding by synthetic PACAP on cerebellar slices from P12 rats treated with vehicle (○) or 1 μg of PACAP (●). Each competition curve was established by quantifying autoradiograms of cerebellar sections incubated with increasing concentrations of PACAP. (Inset) Hill coefficient, _B_max, and IC50 values in control and PACAP-treated animals. Each value is the mean ± SEM of at least three experiments performed in triplicate.

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