Tsai-Feng Fu

Visualizing Long-Term Memory Formation in Two Neurons of the Drosophila Brain

Challenging the Mushroom Bodies Early memory is labile and is gradually consolidated over time into long-lasting, stable memory. In several species, including mammals, memory consolidation depends on protein synthesis. In Drosophila, long-term memory is produced by spaced repetitive training, which induces cyclic adenosine monophosphate (cAMP)–response element–binding protein (CREB)–dependent gene transcription and de novo protein synthesis. Using a large number of genetic tools, Chen et al. (p. 678; see the Perspective by Dubnau) localized this CREB-dependent induction of de novo protein synthesis to two dorsal-anterior-lateral neurons in the adult brain. Importantly, protein synthesis was not required within the mushroom bodies, which are usually considered to be the site of associative learning and memory in insects. Protein synthesis underlying olfactory memory storage takes place outside the mushroom body—the site of memory storage. Long-term memory (LTM) depends on the synthesis of new proteins. Using a temperature-sensitive ribosome-inactivating toxin to acutely inhibit protein synthesis, we screened individual neurons making new proteins after olfactory associative conditioning in Drosophila. Surprisingly, LTM was impaired after inhibiting protein synthesis in two dorsal-anterior-lateral (DAL) neurons but not in the mushroom body (MB), which is considered the adult learning and memory center. Using a photoconvertible fluorescent protein KAEDE to report de novo protein synthesis, we have directly visualized cyclic adenosine monophosphate (cAMP) response element–binding protein (CREB)–dependent transcriptional activation of calcium/calmodulin-dependent protein kinase II and period genes in the DAL neurons after spaced but not massed training. Memory retention was impaired by blocking neural output in DAL during retrieval but not during acquisition or consolidation. These findings suggest an extra-MB memory circuit in Drosophila: LTM consolidation (MB to DAL), storage (DAL), and retrieval (DAL to MB).

Parallel Neural Pathways Mediate CO2 Avoidance Responses in Drosophila

Too Much or Too Little An important task of the nervous system is to distribute information appropriately throughout the brain. The olfactory and gustatory systems of Drosophila provide good models for understanding these processes and the underlying mechanisms (see the Perspective by Su and Carlson). Lin et al. (p. 1338) mapped out the circuit that detects carbon dioxide (CO2), an important environmental and communication signal for fruit flies. Two distinct classes of projection neurons mediate avoidance of high and low concentrations of CO2, while a third class, comprising inhibitory neurons, shuts down the low-concentration pathway at high concentrations. In contrast to other basic taste qualities, salt is innately attractive at low concentrations, but aversive at high concentrations. The mechanisms underlying salt detection are poorly understood in any species mainly because of a lack of specific molecular tools. Zhang et al. (p. 1334) discovered that Drosophila uses two types of gustatory receptor neurons to distinguish between high and low concentrations of salt. One type is activated maximally by low salt and induces attractive feeding behavior. The other class of receptors is activated primarily by high salt and leads to avoidance behavior. Different concentrations of carbon dioxide activate distinct projection neurons and, hence, elicit different responses. [Also see Perspective by Su and Carlson] Different stimulus intensities elicit distinct perceptions, implying that input signals are either conveyed through an overlapping but distinct subpopulation of sensory neurons or channeled into divergent brain circuits according to intensity. In Drosophila, carbon dioxide (CO2) is detected by a single type of olfactory sensory neuron, but information is conveyed to higher brain centers through second-order projection neurons (PNs). Two distinct pathways, PNv-1 and PNv-2, are necessary and sufficient for avoidance responses to low and high CO2 concentrations, respectively. Whereas low concentrations activate PNv-1, high concentrations activate both PNvs and GABAergic PNv-3, which may inhibit PNv-1 pathway-mediated avoidance behavior. Channeling a sensory input into distinct neural pathways allows the perception of an odor to be further modulated by both stimulus intensity and context.

The anti-aging effects of Ludwigia octovalvis on Drosophila melanogaster and SAMP8 mice

We investigated the anti-aging effects of Ludwigia octovalvis (Jacq.) P. H. Raven (Onagraceae), an extract of which is widely consumed as a healthful drink in a number of countries. Using the fruit fly, Drosophila melanogaster, as a model organism, we demonstrated that L. octovalvis extract (LOE) significantly extended fly lifespan on a high, but not a low, calorie diet, indicating that LOE may regulate lifespan through a dietary restriction (DR)-related pathway. LOE also attenuated age-related cognitive decline in both flies and in the senescence-accelerated-prone 8 (SAMP8) mouse, without causing any discernable negative trade-offs, including water intake, food intake, fecundity, or spontaneous motor activity. LOE contained high levels of polyphenols and flavonoids, which possess strong DPPH radical scavenging activity, and was shown to attenuate paraquat-induced oxidative damage and lethality in flies. Gas chromatography–mass spectrometry (GC-MS) analyses identified 17 known molecules, of which β-sitosterol and squalene were the two most abundant. We further demonstrated that β-sitosterol was capable of extending lifespan, likely through activating AMP-activated protein kinase (AMPK) in the fat body of adult flies. Taken together, our data suggest that LOE is a potent anti-aging intervention with potential for treating age-related disorders.

PPL2ab neurons restore sexual responses in aged Drosophila males through dopamine.

Male sexual desire typically declines with ageing. However, our understanding of the neurobiological basis for this phenomenon is limited by our knowledge of the brain circuitry and neuronal pathways controlling male sexual desire. A number of studies across species suggest that dopamine (DA) affects sexual desire. Here we use genetic tools and behavioural assays to identify a novel subset of DA neurons that regulate age-associated male courtship activity in Drosophila. We find that increasing DA levels in a subset of cells in the PPL2ab neuronal cluster is necessary and sufficient for increased sustained courtship in both young and aged male flies. Our results indicate that preventing the age-related decline in DA levels in PPL2ab neurons alleviates diminished courtship behaviours in male Drosophila. These results may provide the foundation for deciphering the circuitry involved in sexual motivation in the male Drosophila brain.

A Single Pair of Neurons Modulates Egg-Laying Decisions in Drosophila

Animals have to judge environmental cues and choose the most suitable option for them from many different options. Female fruit flies selecting an optimum site to deposit their eggs is a biologically important reproductive behavior. When given the direct choice between ovipositing their eggs in a sucrose-containing medium or a caffeine-containing medium, female flies prefer the latter. However, the neural circuits and molecules that regulate this decision-making processes during egg-laying site selection remain poorly understood. In the present study, we found that amnesiac (amn) mutant flies show significant defects in egg-laying decisions, and such defects can be reversed by expressing the wild-type amn transgene in two dorsal paired medial (DPM) neurons in the brain. Silencing neuronal activity with an inward rectifier potassium channel (Kir2.1) in DPM neurons also impairs egg-laying decisions. Finally, the activity in mushroom body αβ neurons is required for the egg-laying behavior, suggesting a possible “DPM-αβ neurons” brain circuit modulating egg-laying decisions. Our results highlight the brain circuits and molecular mechanisms of egg-laying decisions in Drosophila.

Parallel circuits control temperature preference in Drosophila during ageing

The detection of environmental temperature and regulation of body temperature are integral determinants of behaviour for all animals. These functions become less efficient in aged animals, particularly during exposure to cold environments, yet the cellular and molecular mechanisms are not well understood. Here, we identify an age-related change in the temperature preference of adult fruit flies that results from a shift in the relative contributions of two parallel mushroom body (MB) circuits—the β′- and β-systems. The β′-circuit primarily controls cold avoidance through dopamine signalling in young flies, whereas the β-circuit increasingly contributes to cold avoidance as adult flies age. Elevating dopamine levels in β′-afferent neurons of aged flies restores cold sensitivity, suggesting that the alteration of cold avoidance behaviour with ageing is functionally reversible. These results provide a framework for investigating how molecules and individual neural circuits modulate homeostatic alterations during the course of senescence.

Should Adequacy Criteria in Cervicovaginal Cytology Be Modified After Radiotherapy, Chemotherapy, or Hysterectomy?

The general criterion of an unsatisfactory Papanicolaou (Pap) test in the 2001 version of the Bethesda system is not applicable to patients after treatment with radiotherapy, chemotherapy, or hysterectomy. The current study was performed to determine whether specimen adequacy criteria for Pap tests should be modified for these conditions.

Neural circuits for long-term water-reward memory processing in thirsty Drosophila

The intake of water is important for the survival of all animals and drinking water can be used as a reward in thirsty animals. Here we found that thirsty Drosophila melanogaster can associate drinking water with an odour to form a protein-synthesis-dependent water-reward long-term memory (LTM). Furthermore, we found that the reinforcement of LTM requires water-responsive dopaminergic neurons projecting to the restricted region of mushroom body (MB) β′ lobe, which are different from the neurons required for the reinforcement of learning and short-term memory (STM). Synaptic output from α′β′ neurons is required for consolidation, whereas the output from γ and αβ neurons is required for the retrieval of LTM. Finally, two types of MB efferent neurons retrieve LTM from γ and αβ neurons by releasing glutamate and acetylcholine, respectively. Our results therefore cast light on the cellular and molecular mechanisms responsible for processing water-reward LTM in Drosophila.

A Hormone Receptor-Based Transactivator Bridges Different Binary Systems to Precisely Control Spatial-Temporal Gene Expression in Drosophila

The GAL4/UAS gene expression system is a precise means of targeted gene expression employed to study biological phenomena in Drosophila. A modified GAL4/UAS system can be conditionally regulated using a temporal and regional gene expression targeting (TARGET) system that responds to heat shock induction. However heat shock-related temperature shifts sometimes cause unexpected physiological responses that confound behavioral analyses. We describe here the construction of a drug-inducible version of this system that takes advantage of tissue-specific GAL4 driver lines to yield either RU486-activated LexA-progesterone receptor chimeras (LexPR) or β-estradiol-activated LexA-estrogen receptor chimeras (XVE). Upon induction, these chimeras bind to a LexA operator (LexAop) and activate transgene expression. Using GFP expression as a marker for induction in fly brain cells, both approaches are capable of tightly and precisely modulating transgene expression in a temporal and dosage-dependent manner. Additionally, tissue-specific GAL4 drivers resulted in target gene expression that was restricted to those specific tissues. Constitutive expression of the active PKA catalytic subunit using these systems altered the sleep pattern of flies, demonstrating that both systems can regulate transgene expression that precisely mimics regulation that was previously engineered using the GeneSwitch/UAS system. Unlike the limited number of GeneSwitch drivers, this approach allows for the usage of the multitudinous, tissue-specific GAL4 lines for studying temporal gene regulation and tissue-specific gene expression. Together, these new inducible systems provide additional, highly valuable tools available to study gene function in Drosophila.

Diversity and wiring variability of visual local neurons in the Drosophila medulla M6 stratum

Local neurons in the vertebrate retina are instrumental in transforming visual inputs to extract contrast, motion, and color information and in shaping bipolar‐to‐ganglion cell transmission to the brain. In Drosophila, UV vision is represented by R7 inner photoreceptor neurons that project to the medulla M6 stratum, with relatively little known of this downstream substrate. Here, using R7 terminals as references, we generated a 3D volume model of the M6 stratum, which revealed a retinotopic map for UV representations. Using this volume model as a common 3D framework, we compiled and analyzed the spatial distributions of more than 200 single M6‐specific local neurons (M6‐LNs). Based on the segregation of putative dendrites and axons, these local neurons were classified into two families, directional and nondirectional. Neurotransmitter immunostaining suggested a signal routing model in which some visual information is relayed by directional M6‐LNs from the anterior to the posterior M6 and all visual information is inhibited by a diverse population of nondirectional M6‐LNs covering the entire M6 stratum. Our findings suggest that the Drosophila medulla M6 stratum contains diverse LNs that form repeating functional modules similar to those found in the vertebrate inner plexiform layer. J. Comp. Neurol. 522:3795–3816, 2014.