Junjiro Horiuchi

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

Hunger and Memory During starvation, are all brain functions slowed down, or are specific functions disabled to save energy? Plaçais and Preat (p. 440) investigated how the brain of Drosophila deals with severe resource limitation. The brain cut selected expenses to reduce the threat to survival and switched off the formation of aversive long-term memory that depends on costly protein synthesis. However, Hirano et al. (p. 443) focused on mild food-deprivation, which actually enhanced long-term memory formation. Presumably, improved memory should enhance survival when competing for limited food. After longer food deprivation, enhancement of aversive long-term memory decreased, while that of appetitive long-term memory remained high: Presumably, as starvation nears, it becomes more important to pursue food at all costs, and so appetitive memory takes precedence over aversive memories. Different types of memory interact in fed versus starved states to promote survival-oriented behavior. 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.

Mg2+ Block of Drosophila NMDA Receptors Is Required for Long-Term Memory Formation and CREB-Dependent Gene Expression

NMDA receptor (NMDAR) channels allow Ca(2+) influx only during correlated activation of both pre- and postsynaptic cells; a Mg(2+) block mechanism suppresses NMDAR activity when the postsynaptic cell is inactive. Although the importance of NMDARs in associative learning and long-term memory (LTM) formation has been demonstrated, the role of Mg(2+) block in these processes remains unclear. Using transgenic flies expressing NMDARs defective for Mg(2+) block, we found that Mg(2+) block mutants are defective for LTM formation but not associative learning. We demonstrate that LTM-dependent increases in expression of synaptic genes, including homer, staufen, and activin, are abolished in flies expressing Mg(2+) block defective NMDARs. Furthermore, we show that genetic and pharmacological reduction of Mg(2+) block significantly increases expression of a CREB repressor isoform. Our results suggest that Mg(2+) block of NMDARs functions to suppress basal expression of a CREB repressor, thus permitting CREB-dependent gene expression upon LTM induction.

The Drosophila DCO mutation suppresses age-related memory impairment without affecting lifespan

The study of age-related memory impairment (AMI) has been hindered by a lack of AMI-specific mutants. In a screen for such mutants in Drosophila melanogaster, we found that heterozygous mutations of DCO (DCO/+), which encodes the major catalytic subunit of cAMP-dependent protein kinase (PKA), delay AMI more than twofold without affecting lifespan or memory at early ages. AMI is restored when a DCO transgene is expressed in mushroom bodies, structures important for olfactory memory formation. Furthermore, increasing cAMP and PKA activity in mushroom bodies causes premature AMI, whereas reducing activity suppresses AMI. In Drosophila AMI consists of a specific reduction in memory dependent on the amnesiac (amn) gene. amn encodes putative neuropeptides that have been proposed to regulate cAMP levels in mushroom bodies. Notably, both the memory and AMI defects of amn mutants are restored in amn;DCO/+ double mutants, suggesting that AMI is caused by an age-related disruption of amn-dependent memory via PKA activity in mushroom bodies.

The Drosophila cell adhesion molecule Klingon is required for long-term memory formation and is regulated by Notch

The ruslan (rus) mutant was previously identified in a behavioral screen for mutants defective in long-lasting memory, which consists of two consolidated memory types, anesthesia-resistant memory, and protein synthesis-dependent long-term memory (LTM). We demonstrate here that rus is a new allele of klingon (klg), which encodes a homophilic cell adhesion molecule. Klg is acutely required for LTM but not anesthesia-resistant memory formation, and Klg expression increases upon LTM induction. LTM formation also requires activity of the Notch cell-surface receptor. Although defects in Notch have been implicated in memory loss because of Alzheimers disease, downstream signaling linking Notch to memory have not been determined. Strikingly, we found that Notch activity increases upon LTM induction and regulates Klg expression. Furthermore, Notch-induced enhancement of LTM is disrupted by a klg mutation. We propose that Klg is a downstream effector of Notch signaling that links Notch activity to memory.

Calcineurin and Its Regulator Sra/DSCR1 Are Essential for Sleep in Drosophila

Sleep is a fundamental biological process for all animals. However, the molecular mechanisms that regulate sleep are still poorly understood. Here we report that sleep-like behavior in Drosophila is severely impaired by mutations in sarah (sra), a member of the Regulator of Calcineurin (RCAN) family of genes. Sleep reduction in sra mutants is highly correlated with decreases in Sra protein levels. Pan-neural expression of sra rescues this behavioral phenotype, indicating that neuronal sra function is required for normal sleep. Since Sra regulates calcineurin (CN), we generated and examined the behavior of knock-out mutants for all Drosophila CN genes: CanA-14F, Pp2B-14D, and CanA1 (catalytic subunits), and CanB and CanB2 (regulatory subunits). While all mutants show at least minor changes in sleep, CanA-14FKO and CanBKO have striking reductions, suggesting that these are the major CN subunits regulating sleep. In addition, neuronal expression of constitutively active forms of CN catalytic subunits also significantly reduces sleep, demonstrating that both increases and decreases in CN activity inhibit sleep. sra sleep defects are suppressed by CN mutations, indicating that sra and CN affect sleep through a common mechanism. Our results demonstrate that CN and its regulation by Sra are required for normal sleep in Drosophila and identify a critical role of Ca2+/calmodulin-dependent signaling in sleep regulation.

Protein kinase A inhibits a consolidated form of memory in Drosophila

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.

Glial Dysfunction Causes Age-Related Memory Impairment in Drosophila

Several aging phenotypes, including age-related memory impairment (AMI), are thought to be caused by cumulative oxidative damage. In Drosophila, age-related impairments in 1 hr memory can be suppressed by reducing activity of protein kinase A (PKA). However, the mechanism for this effect has been unclear. Here we show that decreasing PKA suppresses AMI by reducing activity of pyruvate carboxylase (PC), a glial metabolic enzyme whose amounts increase upon aging. Increased PC activity causes AMI through a mechanism independent of oxidative damage. Instead, increased PC activity is associated with decreases in D-serine, a glia-derived neuromodulator that regulates NMDA receptor activity. D-serine feeding suppresses both AMI and memory impairment caused by glial overexpression of dPC, indicating that an oxidative stress-independent dysregulation of glial modulation of neuronal activity contributes to AMI in Drosophila.

Mutations in the Drosophila insulin receptor substrate, CHICO, impair olfactory associative learning

CHICO, the Drosophila homolog of vertebrate insulin receptor substrate (IRS), mediates insulin/insulin-like growth factor signaling (IIS), and reductions in chico severely disrupt cell growth and proliferation. We found extensive expression of chico in various Drosophila brain regions including the mushroom bodies (MBs), critical neural structures for olfactory learning. chico null mutants have significantly reduced brain sizes and perform poorly in an olfactory associative learning task, although their sensitivity to the odors and electric shocks used in this learning paradigm are normal. When initial memory is normalized by training for different amounts of time (short-duration training protocols), memory retention and retrieval in chico flies are indistinguishable from that of wild-type flies, demonstrating that chico mutants are defective specifically for memory formation. Inducing expression of a chico(+) transgene in neurons throughout development restores normal learning in a chico background, while inducing chico(+) specifically at the adult stage does not, suggesting that chico is required for development of a brain region required for forming olfactory associations. Significantly, expressing chico(+) in the MBs restores the number of MB neurons to wild-type amounts and also rescues chico learning defects. Our results suggest that chico-dependent growth of the MBs is essential for development of learning ability.

Acute Inhibition of PKA Activity at Old Ages Ameliorates Age-Related Memory Impairment in Drosophila

Age-related memory impairment (AMI) is a critical and debilitating phenotype of brain aging, but its underlying molecular mechanisms are largely unknown. In Drosophila, AMI is highly correlated with PKA activity in the mushroom bodies, neural centers essential for forming associative olfactory memories. Heterozygous mutations in DC0 (DC0/+), which encodes the major catalytic subunit of PKA (PKAc), significantly suppress AMI, while overexpression of a DC0 transgene (DC0+) impairs memory and occludes AMI. PKA activity does not increase upon aging, and it is not clear whether AMI is caused by continual PKA activity throughout aging or by an acute increase in PKA signaling at old ages. Likewise, it is not clear whether AMI can be ameliorated by acute interventions at old ages or whether continuous intervention throughout aging is necessary. We show here that an acute increase in PKA activity at old ages is sufficient to restore normal AMI in DC0/+ flies. Conversely, acute expression of a PKA inhibitory peptide at old ages is sufficient to reverse AMI in a wild-type background. These results indicate that AMI in Drosophila is caused by an age-dependent change in PKA-dependent signaling that can be reversed by acute interventions at old ages.

Shifting transcriptional machinery is required for long-term memory maintenance and modification in Drosophila mushroom bodies

Accumulating evidence suggests that transcriptional regulation is required for maintenance of long-term memories (LTMs). Here we characterize global transcriptional and epigenetic changes that occur during LTM storage in the Drosophila mushroom bodies (MBs), structures important for memory. Although LTM formation requires the CREB transcription factor and its coactivator, CBP, subsequent early maintenance requires CREB and a different coactivator, CRTC. Late maintenance becomes CREB independent and instead requires the transcription factor Bx. Bx expression initially depends on CREB/CRTC activity, but later becomes CREB/CRTC independent. The timing of the CREB/CRTC early maintenance phase correlates with the time window for LTM extinction and we identify different subsets of CREB/CRTC target genes that are required for memory maintenance and extinction. Furthermore, we find that prolonging CREB/CRTC-dependent transcription extends the time window for LTM extinction. Our results demonstrate the dynamic nature of stored memory and its regulation by shifting transcription systems in the MBs.