Spontaneous recovery from extinction depends on the reconsolidation of the acquisition memory in an appetitive learning paradigm in the honeybee (Apis mellifera)

Abstract

Memory retrieval initiates two consolidation processes: consolidation of an extinction memory and reconsolidation of the acquisition memory. The strength of the consolidation processes depends on both the strength of the acquisition memory and the strength of retrieval trials and is correlated with its sensitivity to inhibition. We demonstrate that in the honeybee (Apis mellifera), memory retrieval of a consolidated appetitive olfactory memory leads to both consolidation processes, depending on the number of retrieval trials. Spontaneous recovery from extinction is induced by many (five), but not by few (one and two), retrieval trials. Spontaneous recovery is blocked by emetine, an inhibitor of protein synthesis. We conclude that reconsolidation of the acquisition memory underlies spontaneous recovery.

Figure 1.

In an appetitive, olfactory learning paradigm, one CS-only trial presented after memory is consolidated did not lead to extinction. On day 1, all animals were subjected to three CS-US pairings (A1-A3) with an ITI of 10 min (acquisition). After 24 h, on day 2, one CS-only (C1) trial was presented. At each time point after the CS-only trial, a subgroup of animals was tested with one CS-only trial (retention test). Data from the acquisition phase and the CS-only presentations after 24 h were pooled for all subgroups (retrieved, nonretrieved). For detailed information on performance during acquisition and the CS-only trial of the different subgroups, see supplemental Figure 1-1 A-E (available at www.jneurosci.org as supplemental material). % CR, Percentage of animals that show a proboscis extension response during CS presentation; black circles and bars, retrieved group; gray circles and bars, nonretrieved group. The number in parentheses represents the number of animals in the different subgroups.

Figure 2.

In an appetitive olfactory learning paradigm, two CS-only trials presented after memory is consolidated lead to extinction. On day 1, all animals were subjected to three CS-US pairings (A1-A3) with an ITI of 10 min (acquisition). After 24 h, on day 2, two CS-only (C1, C2) trials were presented with an ITI of 10 min. At each time point after the last CS-only trial, a subgroup of animals was tested with one CS-only trial (retention test). Data of the acquisition phase and the CS-only presentations after 24 h were pooled for all subgroups (retrieved, nonretrieved). For detailed information on performance during acquisition and CS-only trials of the different subgroups, see supplemental Figure 2-1 A-E (available at www.jneurosci.org as supplemental material). Asterisks indicate significant differences (p < 0.05) in the CRs between retrieved and nonretrieved animals of one subgroup. % CR, Percentage of animals that show a proboscis extension response during CS presentation; black circles and bars, retrieved group; gray circles and bars, nonretrieved group. The number in parentheses represents the number of animals in the different subgroups.

Figure 3.

In an appetitive olfactory learning paradigm, five CS-only trials presented after memory is consolidated lead to extinction and spontaneous recovery. On day 1, all animals were subjected to three CS-US pairings (A1-A3) with an ITI of 10 min (acquisition). After 24 h, on day 2, five CS-only trials (C1-C5) were presented with an ITI of 10 min. At each time point after the last CS-only trial, a subgroup of animals was tested with one CS-only trial (retention test). Data from the acquisition phase and the CS-only presentations after 24 h were pooled for all subgroups (retrieved, nonretrieved). For detailed information on performance during acquisition and CS-only trial of the different subgroups, see supplemental Figure 3-1 A-E (available at www.jneurosci.org as supplemental material). Asterisks indicate significant differences (p < 0.05) in the CRs between retrieved and nonretrieved animals of one subgroup. For the retrieved groups, only bars with different letters differ significantly from one another (p < 0.05); nonretrieved groups do not differ from each other. % CR, Percentage of animals that show a proboscis extension response during CS presentation; black circles and bars, retrieved group; gray circles and bars, nonretrieved group. The number in parentheses represents the number of animals in the different subgroups.

Figure 4.

Emetine (Eme; 10 mm) applied systemically 30 min before acquisition blocks long-term memory at 24 h after acquisition. Emetine was injected into the flight muscle 30 min before three CS-US pairings with an ITI of 10 min (acquisition). Memory was tested 24 h after acquisition (retention test), (emetine, n = 57; PBS, n = 61). The asterisk indicates a significant difference between the CRs (p < 0.05). Arrow, Time of injection; gray, PBS-injected control group; black, emetine-injected group; % CR, percentage of animals that show a proboscis extension response during CS presentation.

Figure 5.

Emetine (Eme; 10 mm) applied systemically 30 min before presentation of five CS-only trials blocks spontaneous recovery at a 24 h retention test. On day 1, animals were subjected to three CS-US (A1-A3) pairings with an ITI of 10 min (acquisition). After 24 h, on day 2, emetine was injected into the flight muscle 30 min before five CS-only trials (C1-C5). CS-only trials were presented with an ITI of 10 min. On day 3, memory was tested (retention test). The asterisk indicates a significant difference between the CRs (p < 0.05). A, Retrieved groups (emetine, n = 66; PBS, n = 43). B, Nonretrieved groups (emetine, n = 53; PBS, n = 49). Arrow, Time of injection; gray, PBS-injected control group; black, emetine-injected group; % CR, percentage of animals that showed a proboscis extension response during CS presentation.

Figure 6.

Emetine (Eme; 10 mm) applied systemically 30 min before presentation of two CS-only trials blocks extinction at a 24 h retention test. On day 1, animals were subjected to three CS-US pairings (A1-A3) with an ITI of 10 min (acquisition). After 24 h, on day 2, emetine was injected into the flight muscle 30 min before two CS-only trials (C1,C2) with an ITI of 10 min. On day 3, memory was tested (retention test). The asterisk indicates a significant difference between the CRs (p < 0.05). A, Retrieved groups (emetine, n = 54; PBS, n = 41). B, Nonretrieved groups (emetine, n = 51; PBS, n = 57). Arrow, Time of injection; gray, PBS-injected control group; black, emetine-injected group; % CR, percentage of animals that showed a proboscis extension response during CS presentation.

Figure 7.

Emetine (Eme; 10 mm) applied systemically 30 min before presentation of one CS-only trial does not block extinction or spontaneous recovery at a 24 h retention test. On day 1, animals were subjected to three CS-US pairings (A1-A3) with an ITI of 10 min (acquisition). After 24 h, on day 2, emetine was injected into the flight muscle 30 min before one CS-only trial (C1). On day 3, memory was tested (retention test). The asterisk indicates a significant difference between the CRs (p < 0.05). A, Retrieved groups (emetine, n = 75; PBS, n = 70). B, Control experiment: emetine (10 mm) applied systemically 30 min before acquisition blocks long-term memory at 24 h after acquisition. Emetine was injected into the flight muscle 30 min before three CS-US pairings with an ITI of 10 min (acquisition). Memory was tested 24 h after acquisition (retention test) (emetine, n = 51; PBS, n = 48). Arrow, Time of injection; gray, PBS-injected control group; black, emetine-injected group; % CR, percentage of animals that showed a proboscis extension response during CS presentation.