Fasting Launches CRTC to Facilitate Long-Term Memory Formation in Drosophila

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Science  25 Jan 2013:
Vol. 339, Issue 6118, pp. 443-446
DOI: 10.1126/science.1227170


Canonical aversive long-term memory (LTM) formation in Drosophila requires multiple spaced trainings, whereas appetitive LTM can be formed after a single training. Appetitive LTM requires fasting prior to training, which increases motivation for food intake. However, we found that fasting facilitated LTM formation in general; aversive LTM formation also occurred after single-cycle training when mild fasting was applied before training. Both fasting-dependent LTM (fLTM) and spaced training–dependent LTM (spLTM) required protein synthesis and cyclic adenosine monophosphate response element–binding protein (CREB) activity. However, spLTM required CREB activity in two neural populations—mushroom body and DAL neurons—whereas fLTM required CREB activity only in mushroom body neurons. fLTM uses the CREB coactivator CRTC, whereas spLTM uses the coactivator CBP. Thus, flies use distinct LTM machinery depending on their hunger state.

In Drosophila, canonical aversive long-term memory (LTM), which is dependent on de novo gene expression and protein synthesis, is generated after multiple rounds of spaced training (1, 2). In contrast, appetitive LTM can be formed by single-cycle training (3). Because both aversive and appetitive LTM require protein synthesis (1, 3) and activation of cyclic adenosine monophosphate (cAMP) response element–binding protein (CREB) (35), it is likely that both types of LTM are formed by similar mechanisms. Appetitive and aversive LTM are known to differ [i.e., octopamine is specifically involved in appetitive but not aversive memory formation (6)]. However, it remains unclear why single-cycle training is sufficient for appetitive but not aversive LTM formation. Appetitive LTM cannot form unless fasting precedes training (7). Although fasting increases motivation for food intake—a requirement for appetitive memory (8, 9)—we suspected that fasting may activate a second, motivation-independent, memory mechanism that facilitates LTM formation after single-cycle training.

We deprived flies of food for various periods of time and then subjected them to aversive single-cycle training (Fig. 1A). Fasting prior to training significantly enhanced 1-day memory, with a peak at 16 hours of fasting and a return to nonfasting levels at 20 to 24 hours of fasting (Fig. 1B). In contrast, 16 hours of fasting did not increase short-term memory (STM, measured 1 hour after training) (fig. S1). In this protocol, flies were returned to food vials after training (Fig. 1A), raising a possibility that the perception of food as a reward after training may enhance the previous aversive memory. We tested this possibility by inserting refeeding periods between food deprivation and training. Although fasting followed by a 4-hour refeeding period failed to induce appetitive LTM, it significantly enhanced aversive 1-day memory (fig. S2); this finding suggests that enhancement of aversive memory occurs through a mechanism unrelated to increased motivation or perception of food as a reward. A 6-hour refeeding period was sufficient to prevent aversive memory enhancement. Continuous food deprivation after training suppressed aversive memory enhancement (fig. S3), which indicates that both fasting before training and feeding after training are required to enhance aversive memory.

Fig. 1

Mild fasting facilitates aversive LTM formation after single-cycle training. (A) Schematic diagram of the experimental design; fd, food-deprived. (B) Fasting increases aversive 1-day memory after single-cycle training. Flies were food-deprived for the indicated times before training. (C) CHX treatment prevents fLTM formation. (D) Mild fasting does not enhance 1-day spLTM. (E) MB expression of CREB2-b prevents fLTM formation. (F) CREB2-b expression from a DAL GAL4 driver, G0431, does not affect fLTM. n = 8 to 14 for all data; n.s., not significant (P > 0.05); *P < 0.05, **P < 0.01, ***P < 0.001.

Administration of the protein synthesis inhibitor cycloheximide (CHX) abolished 1-day memory enhancement (Fig. 1C) but had no effect on 1-hour memory (fig. S4), supporting the idea that memory enhancement consists of an increase of LTM. Memory remaining after CHX treatment is likely to be protein synthesis–independent, anesthesia-resistant memory (ARM) (1). Fasting for 16 hours neither enhanced protein synthesis–independent memory (fig. S5) nor canonical aversive LTM generated by spaced training (spLTM) (Fig. 1D). Furthermore, fasting-dependent memory decayed within 4 days, and food deprivation did not enhance 4-day spLTM (fig. S6), indicating that fasting-dependent memory is physiologically different from spLTM.

Fasting-dependent memory was blocked by acute, dose-dependent, expression of CREB2-b, a repressor isoform of CREB (5, 10), in the mushroom bodies (MBs). Expression of the repressor from two copies of UAS-CREB2-b under control of the MB247-Switch (MBsw) GAL4 driver, which induces UAS transgene expression upon RU486 feeding (11), significantly suppressed fasting-dependent memory upon RU486 feeding (Fig. 1E, right panel), whereas expression from one copy of UAS-CREB2-b did not (Fig. 1E, center panel). Defects in LTM formation are highly correlated with CREB2-b amounts (12). We found significantly higher MBsw-dependent expression of CREB proteins in flies carrying two copies of UAS-CREB2-b relative to flies carrying one copy (fig. S7). MBsw-dependent CREB2-b expression did not affect STM in either fed or food-deprived conditions (fig. S8). Because the aversive memory enhanced by fasting is mediated by protein synthesis and CREB, we refer to this memory as fasting-dependent LTM (fLTM). Similar to the results in aversive fLTM, MBsw-dependent CREB2-b expression also decreased appetitive LTM but not appetitive STM (fig. S9).

A recent study (13) concluded that CREB activity in MB neurons is not required for spLTM. In that study, CREB2-b was expressed using the OK107 MB driver and GAL80ts was used to restrict CREB2-b expression to 30°C. However, we found that the GAL80ts construct still inhibited expression of CREB considerably at 30°C (fig. S7). When we acutely expressed higher amounts of CREB2-b in MBs using MBsw, we observed a significant decrease in 1-day spLTM (fig. S10), indicating that CREB activity in the MBs is likely to be required for spLTM.

Consistent with the results of (13), expression of CREB2-b in two dorsal-anterior-lateral (DAL) neurons impaired aversive spLTM (fig. S11). In contrast, expression of CREB2-b in DAL neurons did not affect aversive fLTM (Fig. 1F). Moreover, appetitive LTM was also not affected by expression of CREB2-b in DAL neurons (fig. S12). MBsw did not express GAL4 in DAL neurons (fig. S13).

CREB requires coactivators, including CBP (CREB-binding protein), to activate transcription needed for LTM formation (1416). Acute expression of an inverted repeat of CBP (CBP-IR) (fig. S14A) in MBs significantly impaired spLTM (Fig. 2A) without affecting either STM or 1-day memory after multiple massed trainings, which do not lead to LTM formation (1) (fig. S14, B and C). However, neither aversive fLTM nor appetitive LTM was impaired by CBP-IR expression (Fig. 2B and fig. S14D), indicating that an alternative coactivator may be required for fasting-dependent memory.

Fig. 2

Exclusive role of CBP in spLTM and of CRTC in fLTM. (A and B) Acute expression of CBP-IR in MBs inhibits aversive spLTM (A) but not fLTM (B) formation. (C) Nuclear accumulation of CRTC in MBs after 16 hours of food deprivation. CRTC-HA was coexpressed with nlsGFP [green fluorescent protein (GFP) fused to nuclear localization signal] from a MB GAL driver, MB247. Scale bars, 2 μm. (D) Nuclear intensity of CRTC relative to the dendritic region of MBs; calyx is increased after food deprivation for 16 hours (see fig. S15A). (E and F) Acute CRTC-IR expression in MBs suppresses both aversive (E) and appetitive (F) fLTM formation. (G) Acute CRTC-IR expression in MBs does not affect spLTM formation. n = 8 to 12 for all data; n.s., not significant (P > 0.05); *P < 0.05, **P < 0.01, ***P < 0.001.

Recent studies demonstrate the involvement of a cAMP-regulated transcriptional coactivator (CRTC) in hippocampal plasticity (17, 18). In metabolic tissues, phosphorylated CRTC is sequestered in the cytoplasm while dephosphorylated CRTC translocates to the nucleus (19, 20) to promote CREB-dependent gene expression (2123). Fasting causes CRTC dephosphorylation and activation (24). In line with this, we found significant accumulation of hemagglutinin (HA)–tagged CRTC (CRTC-HA) within MB nuclei after 16 hours of food deprivation (Fig. 2, C and D, and fig. S15A). Subcellular fractionation indicated that food deprivation causes CRTC-HA nuclear translocation without affecting total CRTC-HA amounts (fig. S15C).

To examine the role of CRTC in fLTM and spLTM, we acutely expressed a CRTC inverted repeat (CRTC-IR) (fig. S16A) using MBsw, and observed suppression of aversive fLTM (Fig. 2E) but no effect on STM (fig. S16B). CHX treatment did not further decrease 1-day aversive memory (fig. S16C), and CRTC-IR expression from a second MB driver, OK107, also impaired fLTM formation (fig. S16D). CRTC-IR expression from MBsw also impaired appetitive LTM (Fig. 2F) without affecting appetitive STM (fig. S16E). In contrast, CRTC-IR expression from MBsw did not impair spLTM (Fig. 2G). CRTC-IR expression in DAL neurons had no effect on either aversive fLTM or appetitive LTM (fig. S17). Consistent with our results showing lack of fLTM after 24-hour fasting (Fig. 1B), 1-day aversive memory after 24-hour fasting did not decrease upon CRTC-IR expression in MBs (fig. S16F).

To examine the effects of spaced training on fLTM and the effects of fasting on spLTM, we space-trained fed or fasted flies expressing either CBP-IR or CRTC-IR. When CBP-IR was expressed to impair spLTM, 1-day memory after spaced training was impaired in fed conditions but not in fasting conditions (fig. S18), which suggested that spaced training protocols do not block fLTM. When CRTC-IR was expressed to impair fLTM formation, 1-day memory after spaced training was not affected by fasting (fig. S18), which suggested that mild fasting does not impair spLTM formation.

Is activation of CRTC sufficient to generate fLTM in the absence of fasting? We expressed HA-tagged constitutively active CRTC (CRTC-SA-HA) (24) from MBsw and observed its nuclear accumulation in the absence of fasting (Fig. 3, A and B, and fig. S19). Acute expression of CRTC-SA-HA from MBsw increased 1-day aversive memory after single-cycle training in fed flies, and this increase was not further enhanced by fasting (Fig. 3C). In contrast, expression of control CRTC-HA did not alter the fasting requirement for memory enhancement (Fig. 3D). CRTC-SA-HA expression did not affect feeding itself (fig. S20), which suggested that the memory enhancement is not due to impaired feeding. Taken together, CRTC activity in MBs is necessary and sufficient to form fLTM. Similar to the effects of fasting, CRTC-SA-HA expression did not affect STM (fig. S21) or 4-day spLTM (Fig. 3E).

Fig. 3

Expression of constitutive active CRTC induces aversive fLTM formation in the absence of fasting. (A) CRTC-SA accumulates in MB nuclei, without fasting. Scale bars, 2 μm. (B) Nuclear localization of CRTC-SA, relative to calyx, is increased relative to wild-type CRTC (see fig. S19). (C and D) Acute expression of CRTC-SA-HA (C) but not CRTC-HA (D) in MBs increases 1-day memory after single-cycle training without fasting. (E) Memory enhanced by CRTC-SA-HA decays within 4 days. n = 8 to 10 for all data; n.s., not significant (P > 0.05); *P < 0.05, **P < 0.01, ***P < 0.001.

In mammalian metabolic tissues, CRTC is phosphorylated by insulin signaling, which is suppressed by fasting (21, 24, 25). CRTC phosphorylation is also regulated by insulin signaling in flies (24). To determine whether reduced insulin signaling activates CRTC and promotes fLTM formation, we tested heterozygous mutants for chico, which encodes an adaptor protein required for insulin signaling (26). Although chico1 null mutants are semilethal and defective for olfactory learning, heterozygous chico1/+ mutants are viable and display normal learning (27).

CRTC accumulated in MB nuclei in chico1/+ mutants in the absence of food deprivation (Fig. 4, A and B, and fig. S22). Under conditions where flies were fed, chico1/+ flies had significantly greater 1-day memory after single-cycle training relative to control flies (Fig. 4C), whereas 1-hour memory was unaffected (fig. S23). Enhanced 1-day memory in chico1/+ flies was not further enhanced by fasting (Fig. 4C). Because the chico1/+ mutation does not affect feeding itself (28), the memory enhancement would not seem to be attributable to impaired feeding. The increased 1-day memory in chico1/+ mutants was suppressed by CHX treatment (fig. S24) and CRTC-IR expression using MBsw (Fig. 4D), which suggests that reduced insulin signaling mimics fLTM through activation of CRTC in MBs.

Fig. 4

Reduced insulin signaling induces fLTM formation in the absence of fasting. (A) CRTC-HA accumulates in MB nuclei in chico1/+ mutants. Scale bars, 2 μm. (B) The nuclear localization of CRTC, relative to calyx, is increased in chico1/+ mutants (see fig. S22). (C) The chico1/+ mutation increases aversive 1-day memory after single-cycle training in the absence of food deprivation. (D) Acute CRTC-IR expression in the MBs suppresses the increased aversive 1-day memory of chico1/+ mutants. n = 8 for all data; n.s., not significant (P > 0.05); *P < 0.05, **P < 0.01, ***P < 0.001.

Single-cycle training after mild fasting generates both appetitive and aversive LTM, and CRTC in the MBs plays a key role in both types of LTM. A CRTC-dependent LTM pathway is unlikely to be involved in increasing motivation required to form appetitive memory, because CRTC knockdown did not affect appetitive STM (fig. S16E) and because CRTC-SA expression was not sufficient to form appetitive LTM without prior fasting (fig. S25). Although mild 16-hour fasting induced aversive fLTM, longer 24-hour fasting impaired aversive fLTM (Fig. 1B) but not appetitive LTM (fig. S26). Thus, although aversive and appetitive fLTM share mechanistic similarities, they may be regulated by different inputs controlling motivation and fasting time courses. Because nuclear translocation of CRTC was sustained even after 24 hours of food deprivation (fig. S15, A, B, and E), prolonged fasting may suppress a CRTC-independent step in aversive fLTM formation. spLTM was not affected by 24-hour fasting prior to training (fig. S27), which suggests that the unknown inhibitory effect of 24-hour fasting does not occur after spaced training. Continuous food deprivation after training suppressed aversive fLTM (fig. S3). Elsewhere in this issue, Plaçais and Preat (29) report that continuous food-deprivation after spaced training suppresses spLTM as well.

Suppression of aversive LTM by prolonged fasting may ensure that starving flies pursue available food, with less concern for safety. Although the biological importance of aversive fLTM in natural environments is currently unclear, our results indicate that different physiological states may induce different types of LTM in flies.

Supplementary Materials

Materials and Methods

Figs. S1 to S27

References (3036)

References and Notes

  1. Acknowledgments: We thank M. Montminy, J.Yin, R. Davis, and T. Tabata for materials, and H. Tanimoto and H. Takemori for suggestions. Supported by the Ministry of Education, Culture, Sports, Science, and Technology (MEXT), Japan (grant-in-aid in innovative areas "Systems Molecular Ethology" and KAKENHI grant 21300144) (M.S.); the Japan Science and Technology Agency and PRESTO (Y.H.); and MEXT/Japan Society for the Promotion of Science KAKENHI grants 24700415 and 23700405 (M.M. and K.U.).
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