Phosphorylation of mitogen-activated protein kinase by one-trial and multi-trial classical conditioning

Abstract

The pathway supporting the conditioned stimulus (CS) is one site of plasticity that has been studied extensively in conditioned Hermissenda. Several signal transduction pathways have been implicated in classical conditioning of this preparation, although the major emphasis has been on protein kinase C. Here we provide evidence for the activation and phosphorylation of a mitogen-activated protein kinase (MAPK) pathway by one-trial and multi-trial conditioning. A one-trial in vitro conditioning procedure consisting of light (CS) paired with the application of 5-HT results in the increased incorporation of 32PO4 into proteins detected with two-dimensional gel electrophoresis. Two of the phosphoproteins have molecular weights of 44 and 42 kDa, consistent with extracellular signal-regulated protein kinases (ERK1 and ERK2). Phosphorylation of the 44 and 42 kDa proteins by one-trial conditioning was inhibited by pretreatment with PD098059, A MEK1 (ERK-Activating kinase) inhibitor. Assays of ERK activity with brain myelin basic protein as a substrate revealed greater ERK activity for the group that received one-trial conditioning compared with an unpaired control group. Western blot analysis of phosphorylated ERK using antibodies recognizing the dually phosphorylated forms of ERK1 and ERK2 showed an increase in phosphorylation after one-trial conditioning compared with unpaired controls. The increased phosphorylation of ERK after one-trial conditioning was blocked by pretreatment with PD098059. Hermissenda that received 10 or 15 conditioning trials showed significant behavioral suppression compared with pseudo-random controls. After conditioning and behavioral testing, the conditioned animals showed significantly greater phosphorylation of ERK compared with the pseudo-random controls. These results show that the ERK-MAPK signaling pathway is activated in Pavlovian conditioning of Hermissenda.

Fig. 1.

Phosphate incorporation in 44 and 42 kDa proteins from paired and control groups as analyzed by two-dimensional gel electrophoresis. Laser prints of a storage phosphor screen scan showing³²PO₄ labeling of phosphoproteins after two-dimensional electrophoretic separation. The example shown on thetop is from a group that received one 5 min conditioning trial of light paired with 5-HT (10⁻⁴m). The control group shown on the bottomreceived one 5 min trial of light. An increase in³²PO₄ incorporation was detected for the paired group compared with the controls.

Fig. 2.

Group data (n = 7) showing mean ± SEM E/C ratios of densitometric measurements for the 44 and 42 kDa phosphoproteins after the one-trial conditioning procedure (paired light and 5-HT, 5 min) and the light-only control (5 min). One-trial conditioning resulted in a significant increase in³²PO₄ incorporation for the 44 (mean = 2.4 ± 0.3; p < 0.03) and 42 (mean = 1.8 ± 0.4; p < 0.05) kDa phosphoproteins compared with the control groups.

Fig. 3.

Group data (n = 6) showing mean ± SEM E/C ratios of densitometric measurements for four phosphoproteins examined after one-trial conditioning (paired light and 5-HT, 5 min) and one-trial conditioning for a group pretreated with the MEK1 inhibitor PD098059 (50 μm). Treatment with PD098059 before one-trial conditioning produced a significant reduction in³²PO₄ incorporation into the 44 (mean = 0.59 ± 0.17; p < 0.05) and 42 (mean = 0.34 ± 0.04; p < 0.001) kDa phosphoproteins relative to the conditioned group not treated with PD098059. In contrast, two other phosphoproteins (55 and 29 kDa) in which phosphorylation has been shown to increase with one-trial conditioning were not affected by pretreatment with PD098059.

Fig. 4.

Group data showing mean ± SEM ERK activity measured as phosphorylation of an ERK substrate, brain myelin basic protein. ERK activity for the paired group (mean = 130.7 ± 3; n = 6) that received one trial of light and 5-HT was significantly greater than the unpaired group (mean = 102.7 ± 6.7; n = 3) that received one unpaired trial of light and 5-HT. *p < 0.001.

Fig. 5.

Western blot analysis of phosphorylation and activation of ERK. The preparations were processed for immunoblotting using the chemiluminescence detection method with antibodies recognizing nonphosphorylated and phosphorylated ERK1 and ERK2 or antibodies recognizing the dually phosphorylated forms of ERK1 and ERK2 (Promega). The antibody used for the immunoblotting studies stained a single band on immunoblots, consistent with the molecular weight of ERK. A, After one 10 min trial of light paired with 5-HT (10⁻⁴m), an increased amount of phosphorylated ERK was detected compared with unpaired controls that received 10 min of unpaired light and 5-HT (see Materials and Methods). Neither paired light and 5-HT nor unpaired light and 5-HT changed the total amount of ERK detected with the antibody that recognized both phosphorylated and nonphosphorylated ERK. B, Pretreatment with PD098059 30 min before one-trial conditioning blocks the phosphorylation of ERK detected after conditioning.C, Group data showing mean ± SEM ratios of phospho-ERK/ERK for the groups that received one 10 min trial of light paired with 5-HT (n = 4) and the controls that received one 10 min trial of unpaired light and 5-HT (n = 4). One-trial conditioning resulted in a significant increase in ERK phosphorylation. *p < 0.05.

Fig. 6.

Group data depicting the mean ± SEM suppression ratios for the conditioned group (n = 17) and the pseudo-random controls (n = 15). Conditioning produced a significant behavioral suppression in response to the CS after 10 or 15 conditioning trials. *p < 0.01.

Fig. 7.

Western blot analysis of phosphorylation of ERK after 10 or 15 conditioning trials compared with pseudo-random controls. A, After 10 conditioning trials the amount of phosphorylated ERK was greater than the pseudo-random control group. Neither conditioning nor the control procedure changed the total amount of ERK detected with the antibody that recognized both phosphorylated and nonphosphorylated ERK. B, Group data showing the mean ± SEM ratio of phospho-ERK/ERK for the conditioned group (n = 5) and the pseudo-random controls (n = 5). The conditioned group (mean = 1.6 ± 0.15) showed a significant increase in phosphorylated ERK compared with the controls (mean = 1.3 ± 0.10). *p < 0.02.