殿城 亜矢子

Memory formation and sleep regulation are critical for brain functions in animals from invertebrates to humans. Neuropeptides play a pivotal role in regulating physiological behaviors, including memory formation and sleep. However, the detailed mechanisms by which neuropeptides regulate these physiological behaviors remains unclear. Herein, we report that neuropeptide diuretic hormone 31 (DH31) positively regulates memory formation and sleep in Drosophila melanogaster. The expression of DH31 in the dorsal and ventral fan-shaped body (dFB and vFB) neurons of the central complex and ventral lateral clock neurons (LNvs) in the brain was responsive to sleep regulation. In addition, the expression of membrane-tethered DH31 in dFB neurons rescued sleep defects in Dh31 mutants, suggesting that DH31 secreted from dFB, vFB, and LNvs acts on the DH31 receptor in the dFB to regulate sleep partly in an autoregulatory feedback loop. Moreover, the expression of DH31 in octopaminergic neurons, but not in the dFB neurons, is involved in forming intermediate-term memory. Our results suggest that DH31 regulates memory formation and sleep through distinct neural pathways.

As a normal physiological phenomenon, aging has a significant impact on sleep. Aging leads to sleep impairment, including sleep loss, fragmented sleep, and a lower arousal threshold, leading to various diseases. Because sleep regulates memory consolidation, age-dependent sleep impairment also affects memory. However, the mechanisms underlying age-related sleep dysregulation and its impact on memory remain unclear. Using male and female Drosophila as a model, which possesses sleep characteristics similar to those of mammals and exhibits age-dependent sleep impairment, we performed small-molecule screening to identify novel regulators of age-dependent decline in sleep. The screening identified 3,3'-difluorobenzaldazine (DFB), a positive allosteric modulator of the metabotropic glutamate receptor (mGluR) 5, as a novel sleep-promoting compound in aged flies. We found that mutant flies of mGluR, a single mGluR gene in Drosophila, and decreased mGluR expression had significant impairment in sleep and memory due to olfactory conditioning. The decreased sleep phenotype in the mGluR mutants was not promoted by DFB, suggesting that the effects of DFB on age-dependent sleep impairment are dependent on mGluR. Although aging decreases the expression of mGluR and the binding scaffold proteins Homer and Shank, the transient overexpression of mGluR in neurons improves sleep in both young and aged flies. Overall, these findings indicate that age-dependent decreased expression or function of mGluR impairs sleep and memory in flies, which could lead to age-related sleep and memory impairment.

Age-related changes in the transcriptome lead to memory impairment. Several genes have been identified to cause age-dependent memory impairment (AMI) by changes in their expression, but genetic screens to identify genes critical for AMI have not been performed. The fruit fly is a useful model for studying AMI due to its short lifespan and the availability of consistent techniques and environments to assess its memory ability. We generated a list of candidate genes that act as AMI regulators by performing a comprehensive analysis of RNAsequencing data from young and aged fly heads and genome-wide RNAi screening data to identify memory-regulating genes. A candidate screen using temporal and panneuronal RNAi expression was performed to identify genes critical for AMI. We identified the guanylyl cyclase β-subunit at 100B (gycβ) gene, which encodes a subunit of soluble guanylyl cyclase (sGC), the only intracellular nitric oxide (NO) receptor in fruit flies, as a negative regulator of AMI. RNAi knockdown of gycβ in neurons and NO synthase (NOS) in glia or neurons enhanced the performance of intermediate-term memory (ITM) without apparent effects on memory acquisition. We also showed that pharmacological inhibition of sGC and NOS enhanced ITM in aged individuals, suggesting the possibility that age-related enhancement of the NO-sGC pathway causes memory impairment.

The formation of memory declines with advancing age. However, susceptibility to memory impairments depends on several factors, including the robustness of memory, the responsible neural circuits, and the internal state of aged individuals. How age-dependent changes in internal states and neural circuits affect memory formation remains unclear. Here, we show in Drosophila melanogaster that aged flies of both sexes form robust appetitive memory conditioned with nutritious sugar, which suppresses their high mortality rates during starvation. In contrast, aging impairs the formation of appetitive memory conditioned with non-nutritious sugar that lacks survival benefits for the flies. We found that aging enhanced the preference for nutritious sugar over non-nutritious sugar correlated with an age-dependent increase in the expression of Drosophila neuropeptide F, an ortholog of mammalian neuropeptide Y. Furthermore, a subset of dopaminergic neurons that signal the sweet taste of sugar decreases its function with aging, while a subset of dopaminergic neurons that signal the nutritional value of sugar maintains its function with age. Our results suggest that aging impairs the ability to form memories without survival benefits; however, the ability to form memories with survival benefits is maintained through age-dependent changes in the neural circuits and neuropeptides.SIGNIFICANCE STATEMENT The susceptibility to age-dependent memory impairments depends on the strength of the memory, changes in the responsible neurons, and internal states of aged individuals. How age-dependent changes in such internal states affect neural activity and memory formation remains unclear. We show in Drosophila melanogaster that aged flies of both sexes form robust appetitive memory conditioned with nutritious sugar, which has survival benefits for aged flies. In contrast, aging impairs the formation of appetitive memory conditioned with non-nutritious sugar that lacks survival benefits for the flies. Aging changes the neural circuits including dopamine neurons and neuropeptide F-expressing neurons, leading to the age-dependent impairment in memory with insufficient survival benefits and the preservation of the ability to form memory with survival benefits.

Insulin and insulin-growth-factor-like signaling (IIS) plays important roles in the regulation of development, growth, metabolic homeostasis, and aging, as well as in brain functions such as learning and memory. The temporal-spatial role of IIS in learning and memory and its effect on age-dependent memory impairment remain unclear. Here, we report that intermediate-term memory (ITM), but not short-term memory (STM), in Drosophila aversive olfactory memory requires transient IIS during adulthood. The expression of Drosophila insulin-like peptide 3 (Dilp3) in insulin-producing cells and insulin receptor function in the fat body are essential for ITM. Although the expression of dilp3 decreases with aging, which is unique among dilp genes, the transient expression of dilp3 in aged flies enhances ITM. These findings indicate that ITM is systemically regulated by communication between insulin-producing cells and fat body and that age-dependent changes in IIS contribute to age-related memory impairment.

Although aging is known to impair intermediate-term memory in Drosophila, its effect on protein-synthesis-dependent long-term memory (LTM) is unknown. We show here that LTM is impaired with age, not due to functional defects in synaptic output of mushroom body (MB) neurons, but due to connectivity defects of dorsal paired medial (DPM) neurons with their postsynaptic MB neurons. GFP reconstitution across synaptic partners (GRASP) experiments revealed structural connectivity defects in aged animals of DPM neurons with MB axons in the α lobe neuropil. As a consequence, a protein-synthesis-dependent LTM trace in the α/β MB neurons fails to form. Aging thus impairs protein-synthesis-dependent LTM along with the α/β MB neuron LTM trace by lessening the connectivity of DPM and α/β MB neurons.

How the functional activity of the brain is altered during aging to cause age-related memory impairments is unknown. We used functional cellular imaging to monitor two different calcium-based memory traces that underlie olfactory classical conditioning in young and aged Drosophila. Functional imaging of neural activity in the processes of the dorsal paired medial (DPM) and mushroom body neurons revealed that the capacity to form an intermediate-term memory (ITM) trace in the DPM neurons after learning is lost with age, whereas the capacity to form a short-term memory trace in the α'/β' mushroom body neurons remains unaffected by age. Stimulation of the DPM neurons by activation of a temperature-sensitive cation channel between acquisition and retrieval enhanced ITM in aged but not young flies. These data indicate that the functional state of the DPM neurons is selectively altered with age to cause an age-related impairment of ITM, and demonstrate that altering the excitability of DPM neurons can restore age-related memory impairments.