Learning at different satiation levels reveals parallel functions for the cAMP-protein kinase A cascade in formation of long-term memory

Abstract

Learning and memory formation in intact animals is generally studied under defined parameters, including the control of feeding. We used associative olfactory conditioning of the proboscis extension response in honeybees to address effects of feeding status on processes of learning and memory formation. Comparing groups of animals with different but defined feeding status at the time of conditioning reveals new and characteristic features in memory formation. In animals fed 18 hr earlier, three-trial conditioning induces a stable memory that consists of different phases: a mid-term memory (MTM), translation-dependent early long-term memory (eLTM; 1-2 d), and a transcription-dependent late LTM (lLTM; > or =3 d). Additional feeding of a small amount of sucrose 4 hr before conditioning leads to a loss of all of these memory phases. Interestingly, the basal activity of the cAMP-dependent protein kinase A (PKA), a key player in LTM formation, differs in animals with different satiation levels. Pharmacological rescue of the low basal PKA activity in animals fed 4 hr before conditioning points to a specific function of cAMP-PKA cascade in mediating satiation-dependent memory formation. An increase in PKA activity during conditioning rescues only transcription-dependent lLTM; acquisition, MTM, and eLTM are still impaired. Thus, during conditioning, the cAMP-PKA cascade mediates the induction of the transcription-dependent lLTM, depending on the satiation level. This result provides the first evidence for a central and distinct function of the cAMP-PKA cascade connecting satiation level with learning.

Figure 1.

Associative olfactory learning and memory formation induced by single-trial and three-trial conditioning in bees with different satiation levels. All bees were fed 18 hr before conditioning until satiation. To realize different satiation levels during conditioning, half of the bees received an additional feeding 4 hr before conditioning (fed 4 hr previously). After the first retrieval test 3 hr after training, all animals were fed identically. In animals fed 18 hr earlier, single- (a) and three-trial (b) conditioning (ITI, 2 min) led to acquisition and memory formation as reported in previous studies. Only three-trial conditioning (b) induced a long-lasting memory. The additional feeding 4 hr before conditioning affected acquisition and memory formation in both single- and three-trial-conditioned animals. The asterisks indicate significant differences between the groups (χ² test: *p < 0.02). The number of animals tested in the different groups is indicated in parentheses.

Figure 2.

Inhibition of translation and transcription during conditioning reveals different phases of LTM. Animals were fed 18 hr before conditioning. Thirty minutes before conditioning, animals received an injection (1 μl) of PBS, the transcription blocker actinomycin D (2 mg/ml), or the translation blocker emetine (10 mm). a, Memory induced by single-trial conditioning was affected by neither translation nor transcription blockers. b, Blocking translation or transcription during multiple-trial conditioning specifically impairs distinct phases of LTM. Although blocking transcription during conditioning impairs lLTM (≥ 3 d) (small asterisks), inhibition of translation already affects eLTM (1–2 d) (large asterisks). The number of animals tested in the different groups is indicated in parentheses. Significant differences between the PBS-injected group and the groups injected with drugs are indicated by asterisks (ANOVA repeated measurements and post hoc test; χ² test: *p < 0.02).

Figure 3.

A different satiation status during conditioning affects translation-dependent LTM. To establish different satiation levels during conditioning, half of the bees were fed once, 18 hr before conditioning, whereas the others were fed twice, 18 and 4 hr before conditioning. Thirty minutes before conditioning, animals were injected with 1 μl of PBS or Em/An, a mixture of the translation blockers emetine (Em) (10 mm) and anisomycin (An) (10 mm). Bees were trained with three conditioning trials using a 2 min (a) or a 20 min (b) ITI. Regardless of which translation blockers and ITIs were used, the additional feeding (4 hr before conditioning) affected acquisition and memory at 3 hr, eLTM, and lLTM (open symbols). In the group fed 18 hr earlier, translation blockers led to a clear impairment of eLTM and lLTM independent of the ITI (filled symbols). Asterisks indicate significant differences between the PBS-injected and the Em/An-injected groups fed 18 before conditioning (ANOVA, repeated measurements, and post hoc test: χ² test: *p < 0.01). The number of animals per group is indicated in parentheses.

Figure 4.

Distinguishing effects of satiation status from those of sucrose responsiveness on learning. Sucrose responsiveness of each animal to either 0.1 or 1 m sucrose was tested 15 min before conditioning. The data summarize results of noninjected and PBS-injected animals that received three-trial conditioning (2 and 20 min ITI) (filled circle) and the sorted subgroups (open circle and open triangle) according to their sucrose responsiveness. Although the major subgroups of animals fed 4 hr (a) or 18 hr (b) before conditioning showed the same sucrose responsiveness (0.1 m), they differed in learning and memory formation (a, b, compare open circles). Animals with a reduced sucrose responsiveness (1 m) showed an additional reduction in their performance independent of the satiation status (open triangles). The asterisk in a indicates a significant difference between subgroups with different sucrose responsiveness (ANOVA, repeated measurements). The number of animals per group is indicated in parentheses.

Figure 5.

Feeding status but not sucrose responsiveness affects the cAMP–PKA cascade. Bees were fed according to the behavioral experiments described in Figure 1. Bees were shock-frozen in liquid nitrogen either 4 or 18 hr after the last feeding. a, The data show that the relative basal PKA activity (mean ± SEM) as measured in the central brain and the optical lobes is significantly higher in animals fed 18 hr earlier than in animals fed 4 hr before (t test; *p < 0.01). b, Animals with different sucrose responsiveness (0.1 or 1 m) were selected in the group fed 4 hr previously and compared with animals fed 18 hr earlier (all animals responded to 0.1 m sucrose). Only satiation status, not sucrose responsiveness, affects the basal PKA activity (mean ± SEM) in the brain tissue (t test; *p < 0.01). c, The relative total amount of PKA (mean ± SEM) is similar in animals fed 4 or 18 hr previously, as determined by antibodies against PKA. The number of animals tested for each mean is indicated in the columns.

Figure 6.

Increase in basal PKA activity during conditioning rescues satiation-dependent loss of late LTM. Animals were fed as described in Figure 1. Thirty minutes after injection (1 μl) of PBS or BrcAMP (1 mm) into the hemolymph, bees were used for determining basal PKA activity in the brain (a) or received a three-trial conditioning with an ITI of 2 min (b). a, Values present the mean (±SEM) of the relative basal PKA activity. The number of bees measured for each value is indicated in the columns (t test; *p < 0.05). b, Injection of BrcAMP before conditioning had no effect on learning and memory formation in animals fed 18 hr earlier (filled symbols). Injection of BrcAMP and thus an increase in basal PKA activity during conditioning specifically rescued lLTM in bees fed 4 hr before conditioning (open symbols). The asterisks at days 3 and 4 mark the significant difference between the PBS- and the BrcAMP-injected subgroups (χ² test: *p < 0.01). The number of animals tested in the different groups is indicated in parentheses.

Figure 7.

Memory phases and satiation: dissecting the parallel function of the cAMP–PKA cascade in LTM formation. The diagrams summarize the major molecular mechanisms of memory formation (a) and their modification (b) by the reported interference of satiation on learning. a, All previous experiments used only bees fed 18 hr before conditioning. Single-trial conditioning induces a memory that decays within a few days, whereas multiple-trial conditioning leads to formation of a stable, long-lasting memory. Three-trial conditioning induces different memory phases, including MTM, which requires protein kinase M formed by cleaving protein kinase C during conditioning and an LTM induced in parallel that requires prolonged PKA activation during conditioning. LTM itself can be divided into a translation-dependent eLTM and a transcription-dependent lLTM, both of which are impaired after blocking PKA during conditioning. All of these characteristic memory phases are missing (dotted lines) in bees fed 4 hr before multiple-trial conditioning (b), suggesting an interference of satiation-dependent processes with a site implicated in integrating repeated conditioning trials (dotted area). The specific rescue of lLTM (dark shaded), with still-impaired MTM and eLTM, suggests that two PKA-mediated pathways are required for either eLTM or lLTM induction. Because satiation level also affects single-trial-induced memory, additional molecular interaction sites in addition to the cAMP–PKA pathway must exist between satiation and learning.