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. 2020 Mar 18;318(5):C870–C878. doi: 10.1152/ajpcell.00065.2020

Fig. 2.

Fig. 2.

Pituitary adenylate cyclase activating polypeptide (PACAP)-induced granule cell excitability can be attenuated by MEK and endocytosis inhibitors. A: PACAP-induced increase in excitability remained in slice preparations treated with the adenylyl cyclase inhibitor SQ22536 (20 µM). Effect of drug: F (2,12) = 13.85; P = 0.0009; current: F (9,54) = 50.25; P < 0.0001; and drug × current interaction F (18,108) = 13.63; P < 0.0001. Tukey’s post hoc test exhibited column differences between ACSF condition and SQ22536 + PACAP (P = 0.0010), and between SQ22536 and SQ22536 + PACAP condition (P = 0.0040). B: PACAP-enhanced excitability remained in slice preparations treated with the PLC inhibitor U73122 (20 µM). Effect of drug: F (9,81) = 23.67; P = 0.0101; current: F (9,81) = 23.67; P = <0.0001; and drug × current interaction: F (18,162) = 10.07; P = 0.0001. Tukey’s post hoc multiple-comparisons test exhibited column differences between artificial cerebrospinal fluid (ACSF) and U73122 + PACAP (P = 0.0010), and between U73122 and U73122 + PACAP (P = 0.0133). C: direct adenylyl cyclase activation with forskolin (5 µM) did significantly increase dentate gyrus (DG) granule cell excitability. Effect of forskolin: F (1,8) = 3.145, P = 0.1141, current [F (9, 72) = 28.83, P < 0.0001], and forskolin × current interaction F (1,8) = 3.145; P = 0.005. D: PACAP-induced increase in excitability was essentially eliminated by treatment with the MEK/ERK inhibitor PD98059 (20 µM). Effect of drug F (2,16) = 1.881; P = 0.1846), effect of current F (9,72) = 65.49; P < 0.0001, and current × drug interaction [F (18,144) = 1.784, P = 0.0323]. Tukey’s post hoc test did not show significant group effects. E: PACAP-induced increase in excitability was essentially eliminated by exposure to Pitstop2 (15 µM), an inhibitor of clathrin-mediated endocytosis. Effect of drug F (2,18) = 3.856; P = 0.0404 effect of current F (9,81) = 23.14; P < 0.0001), and drug × current interaction F (18,162) = 3.642; P < 0.0001. Tukey’s post hoc multiple-comparisons test exhibited no significant column differences. F: normalized excitability curves demonstrate that the MEK/ERK inhibitor PD98059 and the clathrin inhibitor Pitstop2 reduced the PACAP-induced increase in excitability by the same extent. For the normalized PACAP excitability curve, the number of action potentials generated by the current steps in control was subtracted from the number of action potentials generated by similar current steps in PACAP. For the inhibitor-normalized excitability curves, the number of action potentials generated by the current steps with only inhibitor present was subtracted from the number of action potentials generated by similar current steps in the presence of inhibitor plus PACAP. The normalized excitability curve for vasoactive intestinal peptide (VIP) is included as a control excitability curve. Data were analyzed with two-way ANOVA with drug and current as repeated measures. ns, not significantly different. *P < 0.05, **P < 0.01; n = 6–10 cells per group. Values are means ± SE.