Pituitary adenylate cyclase-activating polypeptide (PACAP) inhibits the slow afterhyperpolarizing current sIAHP in CA1 pyramidal neurons by activating multiple signaling pathways

Abstract

The slow afterhyperpolarizing current (sIAHP ) is a calcium-dependent potassium current that underlies the late phase of spike frequency adaptation in hippocampal and neocortical neurons. sIAHP is a well-known target of modulation by several neurotransmitters acting via the cyclic AMP (cAMP) and protein kinase A (PKA)-dependent pathway. The neuropeptide pituitary adenylate cyclase activating peptide (PACAP) and its receptors are present in the hippocampal formation. In this study we have investigated the effect of PACAP on the sIAHP and the signal transduction pathway used to modulate intrinsic excitability of hippocampal pyramidal neurons. We show that PACAP inhibits the sIAHP , resulting in a decrease of spike frequency adaptation, in rat CA1 pyramidal cells. The suppression of sIAHP by PACAP is mediated by PAC1 and VPAC1 receptors. Inhibition of PKA reduced the effect of PACAP on sIAHP, suggesting that PACAP exerts part of its inhibitory effect on sIAHP by increasing cAMP and activating PKA. The suppression of sIAHP by PACAP was also strongly hindered by the inhibition of p38 MAP kinase (p38 MAPK). Concomitant inhibition of PKA and p38 MAPK indicates that these two kinases act in a sequential manner in the same pathway leading to the suppression of sIAHP. Conversely, protein kinase C is not part of the signal transduction pathway used by PACAP to inhibit sIAHP in CA1 neurons. Our results show that PACAP enhances the excitability of CA1 pyramidal neurons by inhibiting the sIAHP through the activation of multiple signaling pathways, most prominently cAMP/PKA and p38 MAPK. Our findings disclose a novel modulatory action of p38 MAPK on intrinsic excitability and the sIAHP, underscoring the role of this current as a neuromodulatory hub regulated by multiple protein kinases in cortical neurons.

Keywords: hippocampus; neuropeptide; p38 MAP kinase; protein kinase A; slow afterhyperpolarization.

Figure 1

PACAP-38 and PACAP-27 suppress sI_AHP in hippocampal neurons. Averaged current traces (n = 3) of the sI_AHP before (control) and after application of 500 nM PACAP-38 (A) or 500 nM PACAP-27 (B). Overlaid traces are shown in the rightmost panels (A and B), control (black) and PACAP (grey). The dashed line corresponds to the baseline current. Time course of the effect of PACAP-38 (C) and PACAP-27 (D) on the sI_AHP amplitude from the experiments shown in A and B. Bars indicate the presence of 500 nM PACAP in the bath solution. (E) Effect of 500 nM PACAP-38 (black) and PACAP-27 (white) on sI_AHP amplitude and charge transfer. No significant difference was observed between the two PACAP isoforms. (F) The effect of PACAP-27 on sI_AHP was concentration-dependent. PACAP-27 significantly suppressed sI_AHP amplitude at both 250 nM (n = 18, P < 0.0001) and 500 nM (n = 4, P < 0.01), while at 100 nM PACAP-27 did not cause a significant inhibition of sI_AHP (n = 4, P > 0.05). The difference between the sI_AHP reduction caused by 250 and 500 nM PACAP-27 was not significant (P > 0.05, Dunn's multiple comparisons test). (G) PACAP-27 (250 nM) decreased spike frequency adaptation in a representative CA1 neuron in response to a current injection of 140 pA, resting membrane potential = −59 mV. Similar effects were observed in seven cells.

Figure 2

Selective activation of PAC₁ receptors partly mimics the inhibitory effect of PACAP on the sI_AHP. Averaged current traces (n = 3) of the sI_AHP before (control) and after application of the selective PAC₁ receptor agonist maxadilan at 250 nM (A). In the rightmost panel, the traces in the absence (black) and presence (grey) of maxadilan are superimposed. The dashed line corresponds to the baseline current. (B) Time-course of the effect of 250 nM maxadilan on the amplitude of sI_AHP. Each point is the mean ± SEM of five experiments. Application of maxadilan is indicated by the arrow. (C) 500 nM PACAP-(6–38), a PAC₁ receptor antagonist, prevented the effect of 250 nM maxadilan as shown on averaged current traces (n = 3) of the sI_AHP. The dashed line corresponds to the baseline current. Overlaid traces are shown in the rightmost panel. Bar charts summarizing the reduction of sI_AHP amplitude and charge transfer by 250 nM maxadilan (D) and the absence of maxadilan effect in the presence of 500 nM PACAP-(6–38) (E) in five cells. Error bars indicate S.E.M.

Figure 3

PKA activation contributes to the inhibition sI_AHP by PACAP-27. Averaged current traces (n = 3) of the sI_AHP before and after bath application of 250 nM PACAP-27 in the absence (upper traces) and in the presence (lower traces) of the PKA inhibitor Rp-cAMPS (500 µM) (A). In the rightmost panels, the traces in the absence (black) and presence (grey) of PACAP-27 are superimposed. The dashed line corresponds to the baseline current. (B) Time course of the normalized sI_AHP amplitude from the experiments shown in A before, during, and after PACAP-27 application in the presence (black squares) and absence (white squares) of intracellular Rp-cAMPS. Bar indicates the presence of 250 nM PACAP-27. (C) Bar chart summarizing the effect on the sI_AHP amplitude of PACAP-27 at 250 and 500 nM in the absence (white bars) and presence (black bars) of Rp-cAMPS. Rp-cAMPS significantly reduced the suppression of sI_AHP amplitude by PACAP-27 at 250 nM (P = 0.04, n = 15, Mann–Whitney test), and at 500 nM (P = 0.01, n = 5, unpaired t test). The values for sI_AHP inhibition by PACAP-27 at 250 nM (n = 18) and 500 nM (n = 4) in the absence of Rp-cAMPS are the same as in Figure 1E,F and are reported here for comparison. Error bars indicate S.E.M. * indicates statistical significance.

Figure 4

p38 MAPK activation is involved in the PACAP-27-induced inhibition of sI_AHP. Averaged current traces (n = 3) of the sI_AHP before and after application of 250 nM PACAP-27 in the absence (upper traces) and in the presence (lower traces) of the p38 MAPK inhibitor SB 203580 (20 µM) in the intracellular solution (A). The rightmost panels show superimposed traces in the absence (black) and presence (grey) of PACAP-27. The dashed line corresponds to the baseline current. (B) Time course of the effect of PACAP-27 on the normalized sI_AHP amplitude from the experiments in A, in the absence (white squares) and in the presence (black squares) of SB 203580. Bar indicates the presence of 250 nM PACAP-27. (C) Bar chart summarizing the relative inhibition of the sI_AHP amplitude by 250 nM PACAP-27 under control conditions (n = 18; white bar) and in the presence of SB 203580 (n = 7; black bar). SB 203580 markedly inhibited the suppression of sI_AHP mediated by PACAP-27. Error bars indicate S.E.M. * indicates statistical significance.

Figure 5

Simultaneous inhibition of p38 MAPK and PKA partly prevents the suppression of sI_AHP mediated by PACAP-27. Averaged current traces (n = 3) of the sI_AHP in the absence and presence of 250 nM PACAP-27 recorded without (upper traces) and with (lower traces) the PKA inhibitor Rp-cAMPS (500 µM) and the p38 MAP kinase inhibitor SB 203580 (20 µM) in the patch pipette (A). The rightmost panels display the superimposed traces in the absence (black) and presence (grey) of PACAP-27. The dashed line corresponds to the baseline current. (B) Time course of the effect of PACAP-27 on the sI_AHP amplitude from the experiments in A, with (black squares) and without (white squares) Rp-cAMPS + SB 203580. Bar indicates the application of 250 nM PACAP-27. (C) Bar chart summarizing the relative inhibition of sI_AHP by 250 nM PACAP-27 under control conditions (n = 18; white bar) and in the presence of Rp-cAMPS + SB 203580 (n = 7; black bar). Coapplication of Rp-cAMPS and SB 203580 partly prevented the suppression of sI_AHP amplitude mediated by 250 nM PACAP-27. Error bars indicate S.E.M. * indicates statistical significance.

Figure 6

Activation of EPACs does not suppress sI_AHP in CA1 neurons. Averaged sI_AHP traces (n = 3) recorded in the absence (control) and presence of the EPAC superactivator 8CPT-O-Me-cAMP (5 µM) and of the PKA activator 8CPT-cAMP (5 µM) (A). The rightmost panel shows the same traces superimposed. (B) Time course of action of 8CPT-O-Me-cAMP (5 µM) and 8CPT-cAMP (5 µM) on the sI_AHP amplitude in the same cell as in A. (C) Bar diagram summarizing the effects of 8CPT-O-Me-cAMP (5 µM; black bar) and subsequently applied 8CPT-cAMP (5–50 µM; white bar) on the sI_AHP amplitude in seven cells. The 8CPT-cAMP was used at a concentration of 50 µM in 6 out of 7 cells. The suppression of sI_AHP caused by 8CPT-cAMP was significantly larger than that observed in response to 8CPT-O-Me-cAMP (P < 0.0001, n = 7, unpaired t test). Error bars indicate S.E.M. * indicates statistical significance.

Figure 7

PKC is not involved in the sI_AHP suppression mediated by PACAP-27. Superimposed averaged sI_AHP traces (n = 3) recorded in the absence and presence of 250 nM PACAP-27 without (top panel) and with (lower panel) the PKC inhibitors chelerythrine (20 µM) and BIM-1 (500 nM) in the patch pipette (A). The dashed line corresponds to the baseline current. (B) Time course of the effect of PACAP-27 on the normalized sI_AHP amplitude from the experiments in A, with white squares indicating the absence and black squares the presence of Chelerythrine + BIM-1. Bar indicates the application of 250 nM PACAP-27. (C) Averaged sI_AHP traces (n = 3) recorded in the absence and presence of 250 nM PACAP-27 with chelerythrine (20 µM), BIM-1 (500 nM) and Rp-cAMPS (500 µM) in the patch pipette. The lower panel shows the traces in the absence (black) and presence (grey) of PACAP-27 superimposed. The dashed line represents the baseline current. (D) Bar chart summarizing the relative inhibition of sI_AHP by 250 nM PACAP-27 under control conditions (n = 18; white bar; same data as in Fig. 1F, reported here for comparison) and in the presence of Chelerythrine + BIM-1 (n = 5; black bar), Rp-cAMPS (n = 15; grey bar; same data as in Fig. 3C, reported here for comparison), and Chelerythrine + BIM-1 + Rp-cAMPS (n = 10; stripy bar). Chelerythrine and BIM-1 did not prevent the suppression of sI_AHP by PACAP-27 and did not affect the partial inhibition of the PACAP-27 effect by Rp-cAMPS. Error bars indicate S.E.M. * indicates statistical significance.

Figure 8

Signal transduction pathway mediating the suppression of sI_AHP by PACAP in CA1 pyramidal neurons. Schematic drawing illustrating the signal transduction pathway mediating the effect of PACAP on the sI_AHP in CA1 pyramidal neurons. Pathway components supported by the results obtained in our study are depicted in black; other potential contributors, whose role was discounted by the results of our experiments, are shown in grey. AC, adenylyl cyclase; G_s, G-alpha-s heterotrimeric G-protein; G_q, G-alpha-q heterotrimeric G-protein; PLC, phospholipase C; DAG, diacylglycerol; IP₃, inositol 1,4,5 trisphosphate; MKK, mitogen-activated protein kinase kinase.