Role of hippocampal signaling pathways in long-term memory formation of a nonassociative learning task in the rat

Abstract

Long-term habituation to a novel environment is one of the most elementary forms of nonassociative learning. Here we studied the effect of pre- or posttraining intrahippocampal administration of drugs acting on specific molecular targets on the retention of habituation to a 5-min exposure to an open field measured 24 h later. We also determined whether the exposure to a novel environment resulted in the activation of the same intracellular signaling cascades previously shown to be activated during hippocampal-dependent associative learning. The immediate posttraining bilateral infusion of CNQX (1 microg/side), an AMPA/kainate glutamate receptor antagonist, or of muscimol (0.03 microg/side), a GABA(A) receptor agonist, into the CA1 region of the dorsal hippocampus impaired long-term memory of habituation. The NMDA receptor antagonist AP5 (5 microg/side) impaired habituation when infused 15 min before, but not when infused immediately after, the 5-min training session. In addition, KN-62 (3.6 ng/side), an inhibitor of calcium calmodulin-dependent protein kinase II (CaMKII), was amnesic when infused 15 min before or immediately and 3 h after training. In contrast, the cAMP-dependent protein kinase (PKA) inhibitor Rp-cAMPS, the mitogen-activated protein kinase kinase (MAPKK) inhibitor PD098059, and the protein synthesis inhibitor anisomycin, at doses that fully block memory formation of inhibitory avoidance learning, did not affect habituation to a novel environment. The detection of spatial novelty is associated with a sequential activation of PKA, ERKs (p44 and p42 MAPKs) and CaMKII and the phosphorylation of c-AMP responsive element-binding protein (CREB) in the hippocampus. These findings suggest that memory formation of spatial habituation depends on the functional integrity of NMDA and AMPA/kainate receptors and CaMKII activity in the CA1 region of the hippocampus and that the detection of spatial novelty is accompanied by the activation of at least three different hippocampal protein kinase signaling cascades.

Figure 1

Effect of immediate posttraining intrahippocampal infusions of AP5, CNQX, or muscimol on retention of a spatial habituation in rats. Data are expressed as mean ± SEM of crossings (C) and rearings (R) of training (open bars) and test (closed bars) session performance in an open field with a 24-h interval between sessions. n = 10–14 animals per group. Training-test differences were significant in vehicle and AP5 groups, which indicates that in those two groups there was habituation (t = 4.719, P < 0.001 for vehicle and t = 2.177, P = 0.040 for AP5 group in paired t-test). This was not detectable in groups treated with CNQX and muscimol (indicated with an asterisk).

Figure 2

Effect of 15 min pretraining intrahippocampal infusions of two doses of AP5 on retention of spatial habituation in rats. Data are expressed as mean ± SEM of crossings (C) and rearings (R) of training (open bars) and test (closed bars) session performance in an open field with a 24-h interval between sessions. n = 8–12 animals per group. Training-test differences were significant only in the vehicle group (t = 4.140, P = 0.003 in paired t-test). Habituation was not detectable in groups treated with AP5 in either of the tested doses (indicated with an asterisk).

Figure 3

Effect of pretraining (A: −15 min) or posttraining (B: 0 h; C: 1 h; D: 2 h; E: 3 hr) intrahippocampal infusions of Rp-cAMPS, PD098059, KN-62, or anisomycin on retention of spatial habituation in rats. Data are expressed as mean ± SEM of crossings (C) and rearings (R) of training (open bars) and test (closed bars) session performance in an open field with a 24-h interval between sessions. n = 12–16 animals per group. KN-62 causes retrograde amnesia. All the other treatments were ineffective. Training-test differences were significant in all groups (P < 0.05–0.001, paired Student t-test), except those of KN-62 groups in A, B, and E (t = 18.318, P < 0.001; t = 4.063, P = 0.002; and t = 0.913, P = 0.388, respectively; indicated with an asterisk).

Figure 4

Exposure to a novel environment is associated with the activation of protein kinases and phosphorylation of CREB in the hippocampus at various time intervals (in hours) after a 5-min exposure to an open field. Data are expressed as mean ± SEM in percentage of naive control values. (A) PKA activity, n = 5–6 independent experiments per time point; *** indicates P < 0.001 in Duncan test after ANOVA. (B) Representative immunoblot of phospho αCaMKII and densitometric analysis of five to eight independent experiments per time point; ** indicates P < 0.01 (Dunnet test ). (C, D) Representative immunoblots of phospho p-44 and phospho p-42 (P-p44 and P-p42, respectively) and densitometric analysis of five to 10 independent experiments per time point; * indicates P < 0.05 (Dunnet test). (E) Representative immunoblot of P-CREB and densitometric analysis of four to eight independent experiments per time point; * indicates P < 0.05, ** indicates P < 0.01 (Dunnet test).