Endogenous PACAP acts as a stress response peptide to protect cerebellar neurons from ethanol or oxidative insult

Abstract

The rodent cerebellum is richly supplied with PACAPergic innervation. Exogenous pituitary adenylate cyclase-activating polypeptide (PACAP) increases cerebellar granule cell survival and differentiation in culture, and enhances the number of neuroblasts in the molecular and internal granule cell layers (IGL) when injected postnatally into the cerebellum in vivo. Here, we have investigated the role of endogenous PACAP during cerebellar development by comparing the morphology of normal and PACAP-deficient mouse cerebellum, and the response of cerebellar granule cells from normal and PACAP-deficient mice subjected to neurotoxic insult in culture. There was no difference in cerebellar volume or granule cell number, in 11-day-old wild type versus PACAP-deficient mice. Cultured cerebellar neurons from PACAP-deficient and wild type mice also showed no apparent differences in survival and differentiation either under depolarizing conditions, or non-depolarizing conditions in the presence or absence of either dibutyryl cAMP or 100 nM PACAP. However, cultured cerebellar neurons from PACAP-deficient mice were significantly more sensitive than wild type neurons to ethanol- or hydrogen peroxide-induced toxicity. Differential ethanol toxicity was reversed by addition of 100 nM exogenous PACAP, suggesting that endogenous PACAP has neuroprotective activity in the context of cellular insult or stress. The neuroprotective action of PACAP was mimicked by dibutryl cAMP, indicating that it occurred via activation of adenylate cyclase. These results indicate that PACAP might act to protect the brain from paraphysiological insult, including exposure to toxins or hypoxia.

Fig. 1

Comparison of PACAP wild type versus PACAP-deficient cerebellar development of 11-day-old mice. (A) Quantification of the volume of the cerebellar cortex, as percentage of control (wild type); (B) illustration of the EGL, molecular layer and IGL structure at the level of the Sim lobule of 11-day-old (P11) mice; (C) quantification of the thickness of the EGL, molecular layer and IGL from the Sim of P11 mice; (D) quantification of the density of granule cells in the molecular layer and IGL at the level of the Sim of P11 mice. Each value represents the mean ± S.E.M. of the quantification of seven animals.

Fig. 2

Displacement of [¹²⁵I]-PACAP27 binding sites by synthetic PACAP38 and VIP on 11-day-old mice cerebellar tissue slices. Competition curves were established after quantification of autoradiograms in the external granule cell layer of both wild type and knock-out animals.

Fig. 3

Effect of PACAP on cerebellar granule cell survival. (A) Diagram of the protocol used for the culture and treatment with KCl, dbcAMP, and/or PACAP in the absence of toxic agents, S+ (with serum), S (without serum) (Tables 1 and 2); (B) effect of graded concentrations of−PACAP on granule cell survival from wild type animals. Each value represents the mean (±S.E.M.) of three independent experiments performed in quadruplicate. *P < 0.05 vs. wild type control; **P < 0.01 vs. wild type control.

Fig. 4

Survival of cerebellar granule cells cultured with neurotoxic agents. (A) Diagram of the protocol used for the culture and treatment with ethanol, H₂O₂ or PACAP; (B) effect of graded concentrations of ethanol on granule neurons from wild type and PACAP^−/− animals; (C) effect of graded concentrations of H₂O₂ on granule neurons from wild type and PACAP^−/− animals. Each value represents the mean (±S.E.M.) of three independent experiments performed in quadruplicate. *P< 0.05 vs. wild type; ^#P < 0.05 vs. no PACAP.