Elsevier

Neuroscience

Volume 78, Issue 2, 10 March 1997, Pages 419-430
Neuroscience

Pituitary adenylate cyclase-activating polypeptide promotes cell survival and neurite outgrowth in rat cerebellar neuroblasts

https://doi.org/10.1016/S0306-4522(96)00617-3Get rights and content

Abstract

High concentrations of pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptors have been detected in the rat cerebellum during ontogenesis. In particular, PACAP receptors are actively expressed in immature granule cells, suggesting that PACAP may act as a neurotrophic factor in the developing rat cerebellum. In the present study, we have investigated the effect of PACAP on cell survival and neurite outgrowth in cultured immature cerebellar granule cells. In control conditions, cultured granule cells undergo programmed cell death. Exposure of cultured cells to PACAP for 24 and 48 h provoked a significant increase in the number of living cells. The effect of PACAP on cell survival was inhibited by the PACAP antagonist PACAP(6–38). Vasoactive intestinal polypeptide was approximately 1000 times less potent than PACAP in promoting cell survival. PACAP also induced a significant increase in the number of processes and in the cumulated length of neurites borne by cultured neuroblasts.

The present results demonstrate that PACAP promotes cell survival and neurite outgrowth in cultured immature granule cells. Since PACAP and its receptors are expressed in situ in the rat cerebellar cortex, these data strongly suggest that PACAP plays a physiological role in the survival and differentiation of cerebellar granule cells.

Section snippets

Peptides

PACAP38 and VIP were synthesized by the solid phase methodology described previously.[7]PACAP(6–38) was from the American Peptide Company (Sunnyvale, CA, U.S.A.).

Cell culture

Granule cell suspensions were prepared from cerebelli of eight-day-old Wistar rats, as described previously.[19]Dispersed cells were seeded in multiwell plates (Costar, Cambridge, MA, U.S.A.) coated with poly-l-lysine (5×10−3 M) at a density of 3.5×105 cells per well. The cells were cultured in a chemically defined culture medium

Morphological examination of cultured granule cells

Five hours after plating, cultured granule cells were homogeneously distributed and exhibited a round shape with very few processes (Fig. 1A). Addition of 10−8 M PACAP38 produced no apparent effect on the overall distribution of cells and the number of processes (Fig. 1B). After 24 h, granule cells were still evenly distributed, but their aspect was markedly different (Fig. 1C) as compared to 2-h cultured cells (Fig. 1A). Most round-shaped cells were dying, while surviving cells generally

Discussion

Previous studies have shown the occurrence of substantial amounts of PACAP in the rat cerebellum during development.34, 45It has also been found that PACAP binding sites are expressed by immature granule cells in the rat cerebellar cortex3, 4and that these recognition sites correspond to authentic type I PACAP receptors positively coupled to the adenylyl cyclase and phospholipase C pathways.5, 14The present study has demonstrated that incubation of cultured immature granule cells with PACAP38

Conclusions

The present report indicates that PACAP promotes cell survival and neurite outgrowth in cultured immature granule cells. The neurotrophic activity of PACAP is mediated through high-affinity type I PACAP receptors. These data, together with previous findings showing the presence of PACAP and PACAP receptors in the rat cerebellar cortex, provide strong evidence for the involvement of PACAP in the regulation of apoptosis and differentiation of germinative cells in the developing cerebellum.

Note added in proof

An effect of PACAP on apoptosis of granule cells has been recently reportted by Cavallaro et al., (1996) Am. Soc. Pharm. exp. Ther. 50, 60-66.

Acknowledgements

This work was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM U413), a France–Québec exchange programme (Coopération Scientifique et Technologique Franco-Québécoise) and the Conseil Régional de Haute-Normandie. H.V. is an Affiliated Professor at the Institut National de la Recherche Scientifique-Santé, Montréal.

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