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, 105 (52), 20976-81

Protein Kinase A Inhibits a Consolidated Form of Memory in Drosophila

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Protein Kinase A Inhibits a Consolidated Form of Memory in Drosophila

Junjiro Horiuchi et al. Proc Natl Acad Sci U S A.

Abstract

Increasing activity of the cAMP/protein kinase A (PKA) pathway has often been proposed as an approach to improve memory in various organisms. However, here we demonstrate that single-point mutations, which decrease PKA activity, dramatically improve aversive olfactory memory in Drosophila. These mutations do not affect formation of early memory phases or of protein synthesis-dependent long-term memory but do cause a significant increase in a specific consolidated form of memory, anesthesia-resistant memory. Significantly, heterozygotes of null mutations in PKA are sufficient to cause this memory increase. Expressing a PKA transgene in the mushroom bodies, brain structures critical for memory formation in Drosophila, reduces memory back to wild-type levels. These results indicate that although PKA is critical for formation of several memory phases, it also functions to inhibit at least one memory phase.

Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Fig. 1.
Fig. 1.
Heterozyotes of null (DC0B3/+) and strong (DC0H2/+) DC0 mutations significantly improve memory retention, whereas heterozygotes of weaker DC0 alleles (DC0B10/+ and DC0H3/+) do not. The DC0B3 mutation truncates PKAc by >50 aa, is genetically and phenotypically indistinguishable from deficiency lines lacking DC0, and is likely to be a null mutation in DC0. DC0H2 is classified as a severe or strong DC0 mutation based on the early lethality of hemizyotes. DC0H3, DC0B10, and DC0B12 (data not shown) are classified as medium and weak alleles based on complementation studies and the lethal phase of hemizygotes. Severity of alleles is based on classification by Lane and Kalderon (24). Note that the null allele improves memory to a greater extent than the strong allele, whereas the medium and weak alleles do not improve memory at all. n ≥6 for all data points. DC0B3/+ and DC0H2/+ curves show significant differences compared with wild type with respect to genotype, retention time, and interaction between genotype and retention time as analyzed by 2-way ANOVA (P < 0.003 in all cases). DC0B10/+ and DC0H3/+ curves show no significant differences from wild-type with respect to genotype. Bonferroni post hoc analyses indicate that DC0B3/+ and DC0H2/+ lines do not show significant differences from wild type at retention times of 0 and 1 h but do show significant differences at 3 and 7 h. *, P < 0.05; ***, P < 0.001.
Fig. 2.
Fig. 2.
DC0B3/+ and DC0H2/+ increase ARM. (A) Schematic memory retention curve demonstrating that observed memory retention consists of the sum of an early phase of memory, ASM, and a later phase, ARM. ASM consists of STM and MTM and is gradually consolidated into ARM. (B) Schematic demonstrating that 3-h memory consists of an ASM component and an ARM component. The ARM component can be specifically measured by cold-shocking the flies (cold shock anesthesia) 1 h before testing. Doing so specifically erases the ASM component of memory. ASM can be calculated by subtracting ARM from total 3-h memory. (C) DC0B3/+ and DC0H2/+ significantly improve total 3-h memory (P < 0.01 as assayed by t test). They also improve the cold shock-resistant ARM component of 3-h memory to the same extent (P < 0.05 as assayed by t test). Thus, these DC0 mutations increase ARM and not ASM. n ≥6 for all data points. (D) ARM can also be measured as 24-h memory after 10 massed trainings. ARM after massed training is significantly increased relative to wild type in DC0B3/+ (P < 0.05 as assayed by 1-way ANOVA) and DC0H2/+ (P < 0.05 as assayed by t test), whereas it remains unchanged in DC0B10/+, DC0H3/+, and DC0B12/+ lines. Two separate panels are shown in D because these experiments were performed at different times. Although there is some variability in 24-h memory measured in wild-type flies, both DC0B3/+ and DC0H2/+ always show improved ARM relative to wild-type. n = 5–7. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Fig. 3.
Fig. 3.
DC0/+ mutants do not increase LTM although they do increase ARM in a different genetic background. (A) LTM, a second consolidated form of memory that forms after 10 spaced trainings, is not increased in DC0/+ mutants. LTM measured 4 days after spaced training remains unchanged relative to wild-type in DC0B3/+ and DC0H2/+ flies (P = 0.9629 and P = 0.5730 as assayed by t test). n = 10 and 11, respectively. (B) Memory retention curves of wild-type and DC0B3/+ flies after spaced training. Memory is improved 1 day after training but returns to normal 4 days after training in DC0B3/+ flies. Two-way ANOVA demonstrates significant differences caused by retention time (P < 0.0001) and interaction between genotype and retention time (P = 0.0142). Bonferroni post hoc analyses indicate that DC0B3/+ shows significant differences from wild type at a retention time of 1 day (P < 0.01) but not at 4 and 6 days (P > 0.05). n = 5–10 for each data point. (C and D) Improvement of memory in DC0B3/+ mutants is not caused by specific background effects. Both 3-h memory after single-cycle training (C) and 24-h memory after massed training (D) are improved by the DC0B3/+ mutation in a w1118 background. n ≥ 6. **, P < 0.01; ***, P < 0.001. N.S., not significant.
Fig. 4.
Fig. 4.
A heterozygote of a deficiency line lacking 1 copy of DC0 improves ARM. Expressing PKA in the MBs restores normal memory to a DC0/+ mutant. (A) Total and cold shock-resistant 3-h memory are both increased by similar amounts in a Df(2L7/+ line, which lacks 1 copy of DC0, compared with wild type (P < 0.0001 as assayed by t test). n = 6. (B) Twenty-four-hour memory after massed training is also significantly increased in Df(2L7/+ flies (P = 0.0004 as assayed by t test). n = 14. (C) Expressing a DC0 transgene specifically in the MBs reverts the improved memory of DC0B3/+ back to the wild type. DC0B3/201y and DC0B3/c747 are a DC0B3/+ lines containing the specified MB drivers, 201y and c747, alone. DC0B3/+;PKAc/+ is a DC0B3/+ line containing a DC0 transgene in the absence of a driver, and DC0B3/201y;PKAc/+ and DC0B3/c747;PKAc/+ express the DC0 transgene under 201y and c747 control. One-way ANOVA demonstrates significant differences in 24-h memory caused by genotype in both experiments (P = 0.0045 and P = 0.0002). Bonferroni post hoc analyses show that there are no significant differences between DC0B3/+, DC0B3/201y, DC0B3/c747, and DC0B3/+;PKAc/+ lines (P > 0.05), whereas there are significant differences between DC0B3/+ and wild-type, DC0B3/201y;PKAc/+, and DC0B3/c747;PKAc/+ lines (P < 0.05). n ≥6. *, P < 0.05; ***, P < 0.001; N.S., not significant.
Fig. 5.
Fig. 5.
Expressing PKAc under 201y control does not (A) inhibit ARM in a wild-type background and (B) reduce LTM in a DC0B3/+ background. ARM was measured as 24-h memory after massed training, and LTM was measured as 4-day memory after spaced training. These results indicate that restoration of normal ARM in DC0B3/+ upon 201y-dependent PKAc expression results from complementation of the DC0B3/+ memory phenotype rather than nonspecific memory inhibition because of PKAc overexpression. In both A and B, no significant differences between genotypes were demonstrated when assayed by 1-way ANOVA (P = 0.8983 and P = 0.8516, respectively). n ≥8.

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