Different training procedures recruit either one or two critical periods for contextual memory consolidation, each of which requires protein synthesis and PKA

Abstract

We have used a combined genetic and pharmacological approach to define the time course of the requirement for protein kinase A (PKA) and protein synthesis in long-term memory for contextual fear conditioning in mice. The time course of amnesia in transgenic mice that express R(AB) and have genetically reduced PKA activity in the hippocampus parallels that observed both in mice treated with inhibitors of PKA and mice treated with inhibitors of protein synthesis. This PKA- and protein synthesis-dependent memory develops between 1 hr and 3 hr after training. By injecting the protein synthesis inhibitor anisomycin or the PKA inhibitor Rp-cAMPs at various times after training, we find that depending on the nature of training, contextual memory has either one or two brief consolidation periods requiring synthesis of new proteins, and each of these also requires PKA. Weak training shows two time periods of sensitivity to inhibitors of protein synthesis and PKA, whereas stronger training exhibits only one. These studies underscore the parallel dependence of long-term contextual memory on protein synthesis and PKA and suggest that different training protocols may recruit a common signaling pathway in distinct ways.

Figure 1

The time course of amnesia in ANI and Rp-cAMPs-treated mice parallels that observed in R(AB) transgenic mice. (A) The amnesia for contextual memory in anisomycin-injected mice develops between 1 hr and 3 hr after training. No significant difference in freezing responses between ANI-injected (solid bars) and vehicle-injected (open bars) mice was found 1 hr after training [F(1,10) = 0.32, P = 0.46, n = 6 in each group]. Freezing responses of ANI-injected mice were significantly less than those of vehicle-injected mice at 3 hr [F(1,14) = 14.2, P < 0.001, n = 8 in each group], 6 hr [F(1,14) = 16.7, P < 0.001, n = 8 in each group], and 24 hr after training [F(1, 12) = 18.54, P < 0.001, n = 7 in each group]. (B) The amnesia for contextual memory in Rp-cAMPs-injected mice develops between 1 hr and 3 hr after training. No significant difference in freezing responses between Rp-cAMPs-injected (solid bars) and vehicle-injected (open bars) mice was found 1 hr after training [F(1,14) = 1.28, P = 0.27, n = 8 in each group]. Freezing responses of Rp-cAMPs-injected mice were significantly less than those of vehicle-injected mice at 3 hr [F(1,19) = 13.8, P = 0.001, n = 10 and n = 11 for vehicle- and drug-injected mice, respectively], 6 hr [F(1,12) = 10.3, P < 0.01, n = 7 in each group], and 24 hr after training [F(1,12) = 17.42, P = 0.001, n = 7 in each group]. (C) The amnesia for contextual memory in R(AB) transgenic mice develops between 1 hr and 3 hr after training. No significant difference in freezing responses between R(AB) transgenic (hatched bars) and control (open bars) mice was found 1 hr after training [F(1,10) = 0.49, P = 0.50, n = 6 in each group]. Freezing responses of R(AB) transgenics were significantly less than those of control mice at 3 hr [F(1,31) = 7.82, P < 0.01, n = 14, and n = 19 for controls and mutants, respectively], 6 hr [F(1,12) = 9.01, P = 0.01, n = 6, and n = 8 for controls and mutants, respectively], and 24 hr after training [F(1,12) = 25.74, P < 0.001, n = 7 in each group].

Figure 2

Sensitive periods for protein synthesis and PKA inhibitors following training with 3 CS/US pairings. (A) There is one sensitive time period for the effect of ANI on contextual memory tested at 24 hr. Freezing responses of ANI-injected mice (solid bars) were significantly less than those of vehicle-injected (open bars) animals when ANI was administered at 0 hr [F(1, 19) = 14.57, P < 0.001, n = 10, and n = 11 for vehicle- and ANI-injected animals, respectively]. However, no significant differences in freezing responses between ANI- and vehicle-injected mice were found when injections were made 1 hr [F(1,14) = 0.02, P = 0.86, n = 8 in each group], 4 hr [F(1,16) = 0.42, P = 0.68, n = 9 in each group], 6 hr [F(1,14) = 0.24, P = 0.82, n = 8 in each group], 8 hr [F(1,14) = 0.56, P = 0.59, n = 8 in each group], and 23.5 hr after training [F(1,14) = 0.46, P = 0.64, n = 8 in each group]. (B) There is one sensitive time period for the effect of Rp-cAMPs on contextual memory tested at 24 hr. Freezing responses of Rp-cAMPs-injected mice (shaded bars) were significantly less than those of vehicle-injected animals (open bars) when Rp-cAMPs was administered at 0 hr [F(1,14) = 8.2, P = 0.01, n = 8 in each group]. However, no significant differences in freezing responses between Rp-cAMPs- and vehicle-injected mice were found when injections were made 1 hr [F(1,10) = 0.19, P = 0.67, n = 6 in each group], 4 hr after training [F(1,13) = 0.02, P = 0.87, n = 7, and n = 8 for vehicle and Rp-cAMPs-injected animals, respectively], 6 hr [F(1,10) = 0.05, P = 0.83, n = 6 in each group], 8 hr [F(1,10) = 0.31, P = 0.59, n = 6 in each group], or 23.5 hr after training [F(1,10) = 1.71, P = 0.22, n = 6 in each group].

Figure 3

Sensitive periods for protein synthesis and PKA inhibitors following training with a single CS/US pairing. (A) There are two sensitive time periods for the effect of ANI on contextual memory tested at 24 hr. Freezing responses of ANI-injected mice (solid bars) were significantly less than those of vehicle-injected animals (open bars) when ANI was administered at 0 hr [F(1, 12) = 18.54, P < 0.001, n = 7 in each group; note that data for 0-hr injection and test at 24 hr are also shown in Fig. 1A], or 4 hr after training [F(1, 22) = 8.98, P < 0.01, n = 11, and n = 13 for vehicle- and ANI-injected animals, respectively]. No significant differences in freezing responses between ANI- and vehicle-injected mice were found when injections were made 1 hr [F(1,14) = 0.01, P = 0.90, n = 8 in each group], 6 hr [F(1,14) = 0.01, P = 0.91, n = 8 in each group], 8 hr [F(1,14) = 0.02, P = 0.88, n = 8 in each group], or 23.5 hr after training [F(1,10) = 0.10, P = 0.78, n = 6 in each group]. (B) There are two sensitive time periods for the effect of Rp-cAMPs on contextual memory tested at 24 hr. Freezing responses of Rp-cAMPs-injected mice (shaded bars) were significantly less than those of vehicle-injected animals (open bars) when Rp-cAMPs was administered at 0 hr [F(1,12) = 17.42, P < 0.001, n = 7 in each group; note that data for 0-hr injection and test at 24 hr are also shown in Fig. 1B], or 4 hr after training [F(1, 22) = 7.51, P < 0.01, n = 11, and n = 13 for vehicle- and Rp-cAMPs-injected animals, respectively]. No significant difference in freezing responses between Rp-cAMPs- and vehicle-injected mice was found when injections were made 1 hr [F(1,17) = 0.11, P = 0.74, n = 10, and n = 9 for vehicle- and Rp-cAMPs-injected animals, respectively], 6 hr [F(1,12) = 0.04, P = 0.84, n = 7 in each group], 8 hr [F(1,10) = 0.21, P = 0.66, n = 6 in each group], and 23.5 hr after training [F(1,10) = 0.06, P = 0.82, n = 6 in each group]. (Open bars) Vehicle; (stippled bars) Ro-cAMPS.