Long-term memory (LTM) is stored as functional modifications of relevant neural circuits in the brain. A large body of evidence indicates that the initial establishment of such modifications through the process known as memory consolidation requires learning-dependent transcriptional activation and de novo protein synthesis. However, it remains poorly understood how the consolidated memory is maintained for a long period in the brain, despite constant turnover of molecular substrates. Using the Drosophila courtship conditioning assay of adult males as a memory paradigm, here, we show that in Drosophila, environmental light plays a critical role in LTM maintenance. LTM is impaired when flies are kept in constant darkness (DD) during the memory maintenance phase. Because light activates the brain neurons expressing the neuropeptide pigment-dispersing factor (Pdf), we examined the possible involvement of Pdf neurons in LTM maintenance. Temporal activation of Pdf neurons compensated for the DD-dependent LTM impairment, whereas temporal knockdown of Pdf during the memory maintenance phase impaired LTM in light/dark cycles. Furthermore, we demonstrated that the transcription factor cAMP response element-binding protein (CREB) is required in the memory center, namely, the mushroom bodies (MBs), for LTM maintenance, and Pdf signaling regulates light-dependent transcription via CREB. Our results demonstrate for the first time that universally available environmental light plays a critical role in LTM maintenance by activating the evolutionarily conserved memory modulator CREB in MBs via the Pdf signaling pathway.

Apterous (Ap), the best studied LIM-homeodomain transcription factor in Drosophila, cooperates with the cofactor Chip (Chi) to regulate transcription of specific target genes. Although Ap regulates various developmental processes, its function in the adult brain remains unclear. Here, we report that Ap and Chi in the neurons expressing PDF, a neuropeptide, play important roles in proper sleep/wake regulation in adult flies. PDF-expressing neurons consist of two neuronal clusters: small ventral-lateral neurons (s-LNvs) acting as the circadian pacemaker and large ventral-lateral neurons (l-LNvs) regulating light-driven arousal. We identified that Ap localizes to the nuclei of s-LNvs and l-LNvs. In light-dark (LD) cycles, RNAi knockdown or the targeted expression of dominant-negative forms of Ap or Chi in PDF-expressing neurons or l-LNvs promoted arousal. In contrast, in constant darkness, knockdown of Ap in PDF-expressing neurons did not promote arousal, indicating that a reduced Ap function in PDF-expressing neurons promotes light-driven arousal. Furthermore, Ap expression in l-LNvs showed daily rhythms (peaking at midnight), which are generated by a direct light-dependent mechanism rather than by the endogenous clock. These results raise the possibility that the daily oscillation of Ap expression in l-LNvs may contribute to the buffering of light-driven arousal in wild-type flies.

Memory is initially labile but can be consolidated into stable long-term memory (LTM) that is stored in the brain for extended periods. Despite recent progress, the molecular and cellular mechanisms underlying the intriguing neurobiological processes of LTM remain incompletely understood. Using the Drosophila courtship conditioning assay as a memory paradigm, here, we show that the LIM homeodomain (LIM-HD) transcription factor Apterous (Ap), which is known to regulate various developmental events, is required for both the consolidation and maintenance of LTM. Interestingly, Ap is involved in these 2 memory processes through distinct mechanisms in different neuronal subsets in the adult brain. Ap and its cofactor Chip (Chi) are indispensable for LTM maintenance in the Drosophila memory center, the mushroom bodies (MBs). On the other hand, Ap plays a crucial role in memory consolidation in a Chi-independent manner in pigment dispersing factor (Pdf)-containing large ventral–lateral clock neurons (l-LNvs) that modulate behavioral arousal and sleep. Since disrupted neurotransmission and electrical silencing in clock neurons impair memory consolidation, Ap is suggested to contribute to the stabilization of memory by ensuring the excitability of l-LNvs. Indeed, ex vivo imaging revealed that a reduced function of Ap, but not Chi, results in exaggerated Cl− responses to the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) in l-LNvs, indicating that wild-type (WT) Ap maintains high l-LNv excitability by suppressing the GABA response. Consistently, enhancing the excitability of l-LNvs by knocking down GABAA receptors compensates for the impaired memory consolidation in ap null mutants. Overall, our results revealed unique dual functions of the developmental regulator Ap for LTM consolidation in clock neurons and LTM maintenance in MBs.

In a variety of animal species, females hold a leading position in evaluating potential mating partners. The decision of virgin females to accept or reject a courting male is one of the most critical steps for mating success. In the fruitfly Drosophila melanogaster, however, the molecular and neuronal mechanisms underlying female receptivity are still poorly understood, particularly for virgin females. The Drosophila painless (pain) gene encodes a transient receptor potential (TRP) ion channel. We previously demonstrated that mutations in pain significantly enhance the sexual receptivity of virgin females and that pain expression in painGAL4-positive neurons is necessary and sufficient for pain-mediated regulation of the virgin receptivity. Among the painGAL4-positive neurons in the adult female brain, here we have found that insulin-producing cells (IPCs), a neuronal subset in the pars intercerebralis, are essential in virgin females for the regulation of sexual receptivity through Pain TRP channels. IPC-specific knockdown of pain expression or IPC ablation strongly enhanced female sexual receptivity as was observed in pain mutant females. When pain expression or neuronal activity was conditionally suppressed in adult IPCs, female sexual receptivity was similarly enhanced. Furthermore, both pain mutations and the conditional knockdown of pain expression in IPCs depressed female rejection behaviors toward courting males. Taken together, our results indicate that the Pain TRP channel in IPCs plays an important role in controlling the sexual receptivity of Drosophila virgin females by positively regulating female rejection behaviors during courtship.

In addition to its established function in the regulation of circadian rhythms, the Drosophila gene period (per) also plays an important role in processing long-term memory (LTM). Here, we used courtship conditioning as a learning paradigm and revealed that (1) overexpression and knocking down of per in subsets of brain neurons enhance and suppress LTM, respectively, and (2) suppression of synaptic transmission during memory retrieval in the same neuronal subsets leads to defective LTM. Further analysis strongly suggests that the brain region critical for per-dependent LTM regulation is the fan-shaped body, which is involved in sleep-induced enhancement of courtship LTM.

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17 18 Long-term memory (LTM) is stored as functional modifications of relevant neural circuits 19 in the brain. However, it remains poorly understood how the consolidated memory is 20 maintained for a long period. Here, we show that in Drosophila, universally available 21 environmental light plays a critical role in LTM maintenance by reactivating the memory 22 modulator cAMP response element-binding protein (CREB) via neuropeptide signaling. 23 LTM is impaired when flies are kept in constant darkness during the memory maintenance 24 phase. Because light activates the brain neurons expressing the neuropeptide 25 Pigment-dispersing factor (Pdf) through the brain photoreceptors, we examined the 26 possible involvement of Pdf in LTM maintenance and found that temporal knockdown of 27 Pdf during the memory maintenance phase impairs LTM in light-dark cycles. Furthermore, 28 we demonstrated that the Pdf receptor (Pdfr) and CREB are required in the memory center 29 for LTM maintenance and Pdfr regulates light-dependent transcription via CREB.30 3 90When flies were kept in LL after conditioning, their LTM was intact [ Figure 1A; (7)], as previously 91 reported (Sakai et al., 2004). Furthermore, the LTM of several clock mutants except for period (per) 92 5 mutants is intact (Sakai et al., 2004). Thus, light input, but not the circadian clock, is necessary for LTM 93 maintenance. 94 Sleep plays an important role in the consolidation of Drosophila courtship memory (Donlea et al., 95 2011). However, it remains unclear whether an abnormal sleep phenotype is attributable to disturbed 96 LTM maintenance of flies kept in DD. To examine whether light conditions affect Drosophila sleep (see 97 Materials and methods), sleep amount was measured in flies kept in LD and DD (Figure 2A). The mean 98 amount of daytime sleep of flies kept in DD was lower than that of flies kept in LD (Figures 2A-C).99 However, when flies in LD were slightly deprived of sleep to adjust the amount of daytime sleep to the 100 level of that in DD (Figures 2D-F), their LTM maintenance was not attenuated (Figure 2G). Thus, LTM 101 impairment induced by DD does not simply result from the reduced amount of sleep. 102 103 Expression of brain photoreceptors in Pdf neurons is necessary for LTM maintenance in LD 104 Because light directly activates Pdf neurons through circadian photoreceptors [Rhodopsin 7 (Rh7) and 105 Cryptochrome (Cry)] (Fogle et al., 2011; Ni et al., 2017; Sheeba et al., 2008), Pdf neurons may regulate 106 light-driven LTM maintenance. To examine whether Rh7 and Cry are required for LTM, Rh7 and cry null 107 mutant flies (Rh7 1 and cry 01 , respectively) were used. Since memory on d 1 (one day after conditioning) 108 was intact in Rh7 1 and cry 01 flies after 7 h conditioning (Figure 1B), Rh7 and cry are dispensable for 109 memory consolidation. Unlike the memory on d 1, Rh7 1 and cry 01 flies showed memory impairment on d 110 5 ( Figure 1C), suggesting that Rh7 and Cry are required for keeping LTM more than one day. We next 111 sought to determin...

The neuropeptide pigment-dispersing factor (Pdf) is critically involved in the regulation of circadian rhythms in various insects. The function of Pdf in circadian rhythms has been best studied in the fruitfly, i.e., Drosophila melanogaster. Drosophila Pdf is produced in a small subset of circadian clock neurons in the adult brain and functions as a circadian output signal. Recently, however, Pdf has been shown to play important roles not only in regulating circadian rhythms but also in innate and learned behaviors in Drosophila. In this mini-review, we will focus on the current findings that Pdf signaling and Pdf-producing neurons are essential for consolidating and maintaining long-term memory induced by the courtship conditioning in Drosophila and discuss the mechanisms of courtship memory processing through Pdf-producing neurons.