Context memory formation requires activity-dependent protein degradation in the hippocampus

Abstract

Numerous studies have indicated that the consolidation of contextual fear memories supported by an aversive outcome like footshock requires de novo protein synthesis as well as protein degradation mediated by the ubiquitin-proteasome system (UPS). Context memory formed in the absence of an aversive stimulus by simple exposure to a novel environment requires de novo protein synthesis in both the dorsal (dHPC) and ventral (vHPC) hippocampus. However, the role of UPS-mediated protein degradation in the consolidation of context memory in the absence of a strong aversive stimulus has not been investigated. In the present study, we used the context preexposure facilitation effect (CPFE) procedure, which allows for the dissociation of context learning from context-shock learning, to investigate the role of activity-dependent protein degradation in the dHPC and vHPC during the formation of a context memory. We report that blocking protein degradation with the proteasome inhibitor clasto-lactacystin β-lactone (βLac) or blocking protein synthesis with anisomycin (ANI) immediately after context preexposure significantly impaired context memory formation. Additionally, we examined 20S proteasome activity at different time points following context exposure and saw that the activity of proteasomes in the dHPC increases immediately after stimulus exposure while the vHPC exhibits a biphasic pattern of proteolytic activity. Taken together, these data suggest that the requirement of increased proteolysis during memory consolidation is not driven by processes triggered by the strong aversive outcome (i.e., shock) normally used to support fear conditioning.

Figure 1.

USP-mediated protein degradation and de novo protein synthesis in the dHPC are required for the consolidation of a context representation. (A) Experimental timeline. (B) The diagram shows cannula placement in the CA1 region of the dHPC. The micrograph shows an example placement of dHPC cannula. (C) Average freezing levels during the 5-min context test. Vehicle animals froze significantly more if they were preexposed to the Training context compared to animals that were preexposed to the Control context. Rats preexposed to the Training context and infused with ANI or βLac exhibited impaired freezing during the context test compared to Vehicle animals preexposed to the Training context. (**) P < 0.01.

Figure 2.

USP-mediated protein degradation and de novo protein synthesis in the vHPC are required for the consolidation of a context representation. (A) Experimental timeline. (B) The diagram shows cannula placement in the vHPC. The micrographs show example placements of vHPC cannula. (C) Average freezing levels during the 5-min context test. Vehicle animals froze significantly more if they were preexposed to the Training context compared to animals that were preexposed to the Control context. Rats preexposed to the Training context and infused with ANI or βLac exhibited impaired freezing during the context test compared to Vehicle animals preexposed to the Training context. (**) P < 0.01.

Figure 3.

Proteolytic activity in the dHPC increases immediately and quickly returns to baseline while the vHPC exhibits a biphasic pattern of proteolytic following context exposure. Rats were given 5 min of context exposure, and dHPC and vHPC tissue were collected 0, 30, 60, 90, or 120 min later for Western blotting and in vitro proteasome activity assay. (A) Phosphorylation of Rpt6 normalized to β-actin and expressed as a percentage of the No Exposure (NE) control group. The inset is a representative section of dissected dHPC (light gray) tissue. Phosphorylated Rpt6 marginally increased at 0 and 30 min following context exposure and returned to baseline levels by 60 min. (B) Raw fluorescent units (RFU) for the in vitro 20S proteasome assay for dHPC chymotrypsin, peptidylglutamyl, and trypsin activities expressed as a percentage of the NE control. Chymotrypsin, peptidylglutamyl, and trypsin activities significantly increased immediately following context exposure and returned to baseline by 30 min. (C) Phosphorylation of Rpt6 normalized to β-actin and expressed as a percentage of the No Exposure (NE) control group. The inset is a representative section of dissected vHPC (dark gray) tissue. Phosphorylated Rpt6 significantly increased immediately and following context exposure and returned to baseline by 30 min. (D) Raw fluorescent units (RFU) for the in vitro 20S proteasome assay for vHPC chymotrypsin, peptidylglutamyl, and trypsin activities expressed as a percentage of the NE control. Chymotrypsin and trypsin activities significantly increased immediately following context exposure and returned to baseline by 30 min. Peptidylglutamyl activity significantly increased 90 min following context exposure and returned to baseline by 120 min. (*) P < 0.05, (**) P < 0.01.

Figure 4.

Phosphorylated ERK activity increases immediately following context exposure in the dHPC while increasing 120 min following context in the vHPC. Rats were given 5 min of context exposure and dHPC and vHPC tissue were collected 0, 30, 60, 90, or 120 min later for Western blotting. Phosphorylation of ERK normalized to β-actin and expressed as a percentage of the No Exposure (NE) control group. (A) Phosphorylated ERK activity significantly increased immediately following context exposure and returned to baseline levels by 30 min. (B) Phosphorylated ERK activity significantly increased 120 min following context exposure. (*) P < 0.05, (**) P < 0.01.