The differential role of cortical protein synthesis in taste memory formation and persistence

Abstract

The current dogma suggests that the formation of long-term memory (LTM) is dependent on protein synthesis but persistence of the memory trace is not. However, many of the studies examining the effect of protein synthesis inhibitors (PSIs) on LTM persistence were performed in the hippocampus, which is known to have a time-dependent role in memory storage, rather than the cortex, which is considered to be the main structure to store long-term memories. Here we studied the effect of PSIs on LTM formation and persistence in male Wistar Hola (n ≥ 5) rats by infusing the protein synthesis inhibitor, anisomycin (100 μg, 1 μl), into the gustatory cortex (GC) during LTM formation and persistence in conditioned taste aversion (CTA). We found that local anisomycin infusion to the GC before memory acquisition impaired LTM formation (P = 8.9E - 5), but had no effect on LTM persistence when infused 3 days post acquisition (P = 0.94). However, when we extended the time interval between treatment with anisomycin and testing from 3 days to 14 days, LTM persistence was enhanced (P = 0.01). The enhancement was on the background of stable and non-declining memory, and was not recapitulated by another amnesic agent, APV (10 μg, 1 μl), an N-methyl-d-aspartate receptor antagonist (P = 0.54). In conclusion, CTA LTM remains sensitive to the action of PSIs in the GC even 3 days following memory acquisition. This sensitivity is differentially expressed between the formation and persistence of LTM, suggesting that increased cortical protein synthesis promotes LTM formation, whereas decreased protein synthesis promotes LTM persistence.

Figure 1

Stereotactic infusion of anisomycin into the gustatory cortex (GC) before acquisition impairs long-term memory, whereas infusing it 3 days later has no effect. (a) Anisomycin (n=6) infusion to the GC 20 min before conditioned taste aversion (CTA) results in a lower aversion index (AI) than in vehicle-infused (n=6) rats. **P=8.9E−5. (b) One week following the completion of CTA for saccharin both groups (n=6) were subjected to CTA for a different and novel taste (0.3% NaCl), which resulted in higher aversion toward NaCl with no significant difference between them. (c) Anisomycin (n=9) or vehicle (n=11) were infused to the gustatory cortex 3 days following CTA acquisition. Both groups showed high aversion with no significant difference between them when tested 4 days later. (d) On continued testing for four more consecutive days both groups acquired extinction to saccharin (which is expressed as a significant difference between day1 and day5) with no significant difference between them. ^# P=0.845.

Figure 2

Injection of anisomycin into the gustatory cortex (GC) before weak conditioned taste aversion (CTA) protocol impairs long-term memory, whereas infusing it 3 days later has no effect. (a) Weak CTA protocol: reducing lithium chloride (LiCl) concentration from 0.15 mol/l to 0.05 mol/l or 0.03 mol/l during CTA training results in lower aversion index (AI) when tested 3 days later, thus allowing detection of both enhancement and impairment of memory. 0.15 mol/l, n=15; 0.05 mol/l, n=5; 0.03 mol/l, n=9. Main effect of group, analysis of variance (ANOVA), ^# P=0.0001. *P=0.026 between 0.15 mol/l and 0.05 mol/l. P=0.047 between 0.05 mol/l and 0.03 mol/l. (b) Weak CTA long-term memory (LTM) formation is sensitive to protein synthesis inhibition. Animals were trained with 0.05 mol/l of LiCl (weak CTA protocol) 20 min after anisomycin or vehicle injection to the GC. Testing the animals 3 days later resulted in memory impairment (Figure 4e: n=8 per group, t₁₅=12.6, **P=0.0001, t test), demonstrating that the sensitivity of LTM to protein synthesis inhibition before memory acquisition applies for weak CTA protocol as well. (c) Anisomycin (n=9) or vehicle (n=13) were infused to the gustatory cortex 3 days following weak CTA acquisition. Both groups showed lower aversion than standard CTA (see text) with no significant difference between them when tested 4 days later. (d) On continued testing for two more consecutive days both groups acquired extinction for saccharin aversiveness (which is expressed as a significant difference between day1 and day3) with no significant difference between them ^# P=0.474.

Figure 3

Anisomycin infusion to the gustatory cortex reduces spine density and length. Anisomycin or vehicle was infused to the rat gustatory cortex and brains were subjected to Golgi staining 6 days later. Anisomycin-infused animals showed significant reduction both in spine density (**P=0.0017, t=3.299, degrees of freedom (d.f.)=57) and spine length (*P=0.0336, t=2.141, d.f.=56). The analysis is based on N=28–32, where N=analyzed neuron, taken from four animals in each condition.

Figure 4

Increasing the time interval between anisomycin infusion to memory testing results in long-term memory enhancement. (a) Anisomycin (n=14) or vehicle (n=16) was infused to the rat gustatory cortex 3 days following weak conditioned taste aversion (CTA) acquisition. When tested 14 days later, animals receiving anisomycin showed significantly higher aversion toward saccharin than animals receiving saline vehicle infusion. **P=0.01. (b) On continued testing for two additional consecutive days both groups acquired extinction of aversion to saccharin (**P=0.001 for anisomycin, **P=0.005 for vehicle) with no significant difference between them. ^# P=0.182. (c) One week following the completion of CTA for saccharin both groups (only last batch of animals. n=5) were subjected to CTA for a different and novel taste (0.3% NaCl) which resulted in aversion toward NaCl with no significant difference between the groups. (d) Aversion to saccharin in naive rats is not influenced when tested 14 days following infusion of anisomycin to the gustatory cortex (n=15) as assessed by comparison to vehicle-infused rats (n=18).

Figure 5

Memory enhancement is on the background of a stable and non-declining memory and is not susceptible to other interruptions to memory consolidation. (a) There is no significant difference between groups of animals that were tested 1, 3, and 21 days following weak conditioned taste aversion (CTA) (1 day n=15, 3 days n=11, 21 days n=14; Kruskal–Wallis test: χ²=0.016; P=0.99). (b) APV (n=11) or vehicle (n=12) was infused to the gustatory cortex (GC) 3 days following CTA acquisition. Both groups showed aversion with no significant difference between them when tested 4 days later (t test: t₂₁=0.63, P=0.54). (c) Anisomycin injection to the GC has no effect on 0.1% saccharin consumption. Animals were injected with anisomycin (n=6) or vehicle (n=8) to the GC 20 min before 0.1% saccharin consumption. There was no significant difference between the groups (P=0.78).