RESUMO
Calcium imaging with protein-based indicators1,2 is widely used to follow neural activity in intact nervous systems, but current protein sensors report neural activity at timescales much slower than electrical signalling and are limited by trade-offs between sensitivity and kinetics. Here we used large-scale screening and structure-guided mutagenesis to develop and optimize several fast and sensitive GCaMP-type indicators3-8. The resulting 'jGCaMP8' sensors, based on the calcium-binding protein calmodulin and a fragment of endothelial nitric oxide synthase, have ultra-fast kinetics (half-rise times of 2 ms) and the highest sensitivity for neural activity reported for a protein-based calcium sensor. jGCaMP8 sensors will allow tracking of large populations of neurons on timescales relevant to neural computation.
Assuntos
Sinalização do Cálcio , Cálcio , Calmodulina , Neurônios , Óxido Nítrico Sintase Tipo III , Fragmentos de Peptídeos , Cálcio/análise , Cálcio/metabolismo , Calmodulina/metabolismo , Neurônios/metabolismo , Cinética , Óxido Nítrico Sintase Tipo III/química , Óxido Nítrico Sintase Tipo III/metabolismo , Fatores de Tempo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismoRESUMO
Femtosecond lasers at fixed wavelengths above 1,000 nm are powerful, stable and inexpensive, making them promising sources for two-photon microscopy. Biosensors optimized for these wavelengths are needed for both next-generation microscopes and affordable turn-key systems. Here we report jYCaMP1, a yellow variant of the calcium indicator jGCaMP7 that outperforms its parent in mice and flies at excitation wavelengths above 1,000 nm and enables improved two-color calcium imaging with red fluorescent protein-based indicators.
Assuntos
Cálcio/análise , Corantes Fluorescentes/química , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Animais , Drosophila , Feminino , Lasers , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Imagem Molecular , Córtex Somatossensorial/químicaRESUMO
A P-type H+-ATPase is the primary transporter that converts ATP to electrochemical energy at the plasma membrane of higher plants. Its product, the proton-motive force, is composed of an electrical potential and a pH gradient. Many studies have demonstrated that this proton-motive force not only drives the secondary transporters required for nutrient uptake, but also plays a direct role in regulating cell expansion. Here, we have generated a transgenic Arabidopsis (Arabidopsis thaliana) plant expressing H+-ATPase isoform 2 (AHA2) that is translationally fused with a fluorescent protein and examined its cellular localization by live-cell microscopy. Using a 3D imaging approach with seedlings grown for various times under a variety of light intensities, we demonstrate that AHA2 localization at the plasma membrane of root cells requires light. In dim light conditions, AHA2 is found in intracellular compartments, in addition to the plasma membrane. This localization profile was age-dependent and specific to cell types found in the transition zone located between the meristem and elongation zones. The accumulation of AHA2 in intracellular compartments is consistent with reduced H+ secretion near the transition zone and the suppression of root growth. By examining AHA2 localization in a knockout mutant of a receptor protein kinase, FERONIA, we found that the intracellular accumulation of AHA2 in the transition zone is dependent on a functional FERONIA-dependent inhibitory response in root elongation. Overall, this study provides a molecular underpinning for understanding the genetic, environmental, and developmental factors influencing root growth via localization of the plasma membrane H+-ATPase.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Arabidopsis/genética , Membrana Celular/enzimologia , Meio Ambiente , ATPases Translocadoras de Prótons/metabolismo , Arabidopsis/efeitos da radiação , Membrana Celular/efeitos da radiação , Corantes Fluorescentes/metabolismo , Concentração de Íons de Hidrogênio , Espaço Intracelular/metabolismo , Luz , Especificidade de Órgãos , Fosfotransferases/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Raízes de Plantas/efeitos da radiação , Proteínas Recombinantes de Fusão/metabolismo , Plântula/crescimento & desenvolvimento , Plântula/efeitos da radiaçãoRESUMO
Sleep in Drosophila shares many features with mammalian sleep, but it remains unknown whether spontaneous and evoked activity of individual neurons change with the sleep/wake cycle in flies as they do in mammals. Here we used calcium imaging to assess how the Kenyon cells in the fly mushroom bodies change their activity and reactivity to stimuli during sleep, wake, and after short or long sleep deprivation. As before, sleep was defined as a period of immobility of >5 min associated with a reduced behavioral response to a stimulus. We found that calcium levels in Kenyon cells decline when flies fall asleep and increase when they wake up. Moreover, calcium transients in response to two different stimuli are larger in awake flies than in sleeping flies. The activity of Kenyon cells is also affected by sleep/wake history: in awake flies, more cells are spontaneously active and responding to stimuli if the last several hours (5-8 h) before imaging were spent awake rather than asleep. By contrast, long wake (≥29 h) reduces both baseline and evoked neural activity and decreases the ability of neurons to respond consistently to the same repeated stimulus. The latter finding may underlie some of the negative effects of sleep deprivation on cognitive performance and is consistent with the occurrence of local sleep during wake as described in behaving rats. Thus, calcium imaging uncovers new similarities between fly and mammalian sleep: fly neurons are more active and reactive in wake than in sleep, and their activity tracks sleep/wake history.
Assuntos
Cálcio/metabolismo , Drosophila melanogaster/fisiologia , Neurônios/fisiologia , Sono/fisiologia , Vigília/fisiologia , Ácido Acético/farmacologia , Animais , Animais Geneticamente Modificados , Calmodulina/genética , Calmodulina/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Lasers , Microscopia de Fluorescência por Excitação Multifotônica , Corpos Pedunculados/citologia , Corpos Pedunculados/metabolismo , Corpos Pedunculados/fisiologia , Quinase de Cadeia Leve de Miosina/genética , Quinase de Cadeia Leve de Miosina/metabolismo , Neuroimagem/métodos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Oxigênio/farmacologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Privação do Sono/fisiopatologiaRESUMO
Animals are often bombarded with visual information and must prioritize specific visual features based on their current needs. The neuronal circuits that detect and relay visual features have been well-studied. Yet, much less is known about how an animal adjusts its visual attention as its goals or environmental conditions change. During social behaviors, flies need to focus on nearby flies. Here, we study how the flow of visual information is altered when female Drosophila enter an aggressive state. From the connectome, we identified three state-dependent circuit motifs poised to selectively amplify the response of an aggressive female to fly-sized visual objects: convergence of excitatory inputs from neurons conveying select visual features and internal state; dendritic disinhibition of select visual feature detectors; and a switch that toggles between two visual feature detectors. Using cell-type-specific genetic tools, together with behavioral and neurophysiological analyses, we show that each of these circuit motifs function during female aggression. We reveal that features of this same switch operate in males during courtship pursuit, suggesting that disparate social behaviors may share circuit mechanisms. Our work provides a compelling example of using the connectome to infer circuit mechanisms that underlie dynamic processing of sensory signals.
RESUMO
Dopaminergic neurons with distinct projection patterns and physiological properties compose memory subsystems in a brain. However, it is poorly understood whether or how they interact during complex learning. Here, we identify a feedforward circuit formed between dopamine subsystems and show that it is essential for second-order conditioning, an ethologically important form of higher-order associative learning. The Drosophila mushroom body comprises a series of dopaminergic compartments, each of which exhibits distinct memory dynamics. We find that a slow and stable memory compartment can serve as an effective 'teacher' by instructing other faster and transient memory compartments via a single key interneuron, which we identify by connectome analysis and neurotransmitter prediction. This excitatory interneuron acquires enhanced response to reward-predicting odor after first-order conditioning and, upon activation, evokes dopamine release in the 'student' compartments. These hierarchical connections between dopamine subsystems explain distinct properties of first- and second-order memory long known by behavioral psychologists.
Assuntos
Dopamina , Drosophila , Animais , Drosophila/fisiologia , Aprendizagem , Encéfalo , Odorantes , Neurônios Dopaminérgicos/fisiologia , Corpos Pedunculados/fisiologia , Drosophila melanogaster/fisiologia , Olfato/fisiologiaRESUMO
How memories are used by the brain to guide future action is poorly understood. In olfactory associative learning in Drosophila, multiple compartments of the mushroom body act in parallel to assign a valence to a stimulus. Here, we show that appetitive memories stored in different compartments induce different levels of upwind locomotion. Using a photoactivation screen of a new collection of split-GAL4 drivers and EM connectomics, we identified a cluster of neurons postsynaptic to the mushroom body output neurons (MBONs) that can trigger robust upwind steering. These UpWind Neurons (UpWiNs) integrate inhibitory and excitatory synaptic inputs from MBONs of appetitive and aversive memory compartments, respectively. After formation of appetitive memory, UpWiNs acquire enhanced response to reward-predicting odors as the response of the inhibitory presynaptic MBON undergoes depression. Blocking UpWiNs impaired appetitive memory and reduced upwind locomotion during retrieval. Photoactivation of UpWiNs also increased the chance of returning to a location where activation was terminated, suggesting an additional role in olfactory navigation. Thus, our results provide insight into how learned abstract valences are gradually transformed into concrete memory-driven actions through divergent and convergent networks, a neuronal architecture that is commonly found in the vertebrate and invertebrate brains.
Assuntos
Aprendizagem , Vento , Animais , Drosophila/fisiologia , Olfato/fisiologia , Neurônios/fisiologia , Corpos Pedunculados/fisiologia , Drosophila melanogaster/fisiologiaRESUMO
Most of us sleep 7-8 h per night, and if we are deprived of sleep our performance suffers greatly; however, a few do well with just 3-4 h of sleep-a trait that seems to run in families. Determining which genes underlie this phenotype could shed light on the mechanisms and functions of sleep. To do so, we performed mutagenesis in Drosophila melanogaster, because flies also sleep for many hours and, when sleep deprived, show sleep rebound and performance impairments. By screening 9,000 mutant lines, we found minisleep (mns), a line that sleeps for one-third of the wild-type amount. We show that mns flies perform normally in a number of tasks, have preserved sleep homeostasis, but are not impaired by sleep deprivation. We then show that mns flies carry a point mutation in a conserved domain of the Shaker gene. Moreover, after crossing out genetic modifiers accumulated over many generations, other Shaker alleles also become short sleepers and fail to complement the mns phenotype. Finally, we show that short-sleeping Shaker flies have a reduced lifespan. Shaker, which encodes a voltage-dependent potassium channel controlling membrane repolarization and transmitter release, may thus regulate sleep need or efficiency.
Assuntos
Drosophila melanogaster/metabolismo , Mutação Puntual/genética , Canais de Potássio/metabolismo , Privação do Sono/genética , Privação do Sono/fisiopatologia , Sequência de Aminoácidos , Animais , Comportamento Animal/fisiologia , Ritmo Circadiano/genética , Ritmo Circadiano/fisiologia , Sequência Conservada , Cruzamentos Genéticos , Escuridão , Proteínas de Drosophila , Drosophila melanogaster/genética , Feminino , Genes Recessivos/genética , Teste de Complementação Genética , Homeostase , Humanos , Luz , Longevidade/genética , Longevidade/fisiologia , Masculino , Mamíferos/fisiologia , Dados de Sequência Molecular , Atividade Motora/fisiologia , Fenótipo , Canais de Potássio/química , Canais de Potássio/genética , Estrutura Terciária de Proteína , Superfamília Shaker de Canais de Potássio , Sono/genética , Sono/fisiologia , Fatores de Tempo , Cromossomo X/genéticaRESUMO
Sleep need is affected by developmental stage and neuronal plasticity, but the underlying mechanisms remain unclear. The fragile X mental retardation gene Fmr1, whose loss-of-function mutation causes the most common form of inherited mental retardation in humans, is involved in synaptogenesis and synaptic plasticity, and its expression depends on both developmental stage and waking experience. Fmr1 is highly conserved across species and Drosophila mutants carrying dFmr1 loss-of-function or gain-of-function mutations are well characterized: amorphs have overgrown dendritic trees with larger synaptic boutons, developmental defects in pruning, and enhanced neurotransmission, while hypermorphs show opposite defects, including dendritic and axonal underbranching and loss of synapse differentiation. We find here that dFmr1 amorphs are long sleepers and hypermorphs are short sleepers, while both show increased locomotor activity and shortened lifespan. Both amorphs and hypermorphs also show abnormal sleep homeostasis, with impaired waking performance and no sleep rebound after sleep deprivation. An impairment in the circadian regulation of sleep cannot account for the altered sleep phenotype of dFmr1 mutants, nor can an abnormal activation of glutamatergic metabotropic receptors. Moreover, overexpression of dFmr1 throughout the mushroom bodies is sufficient to reduce sleep. Finally, dFmr1 protein levels are modulated by both developmental stage and behavioral state, with increased expression immediately after eclosure and after prolonged wakefulness. Thus, dFmr1 expression dose-dependently affects both sleep and synapses, suggesting that changes in sleep time in dFmr1 mutants may derive from changes in synaptic physiology.
Assuntos
Encéfalo/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteína do X Frágil da Deficiência Intelectual/genética , Sono/genética , Transmissão Sináptica/genética , Animais , Encéfalo/anormalidades , Encéfalo/fisiopatologia , Diferenciação Celular/genética , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Longevidade/genética , Atividade Motora/genética , Corpos Pedunculados/anormalidades , Corpos Pedunculados/metabolismo , Corpos Pedunculados/fisiopatologia , Plasticidade Neuronal/genética , Receptores de Glutamato Metabotrópico/genética , Privação do Sono/genética , Privação do Sono/metabolismo , Privação do Sono/fisiopatologia , Vigília/genéticaRESUMO
BACKGROUND: Epidemiological studies in humans suggest that a decrease in daily sleep duration is associated with reduced lifespan, but this issue remains controversial. Other studies in humans also show that both sleep quantity and sleep quality decrease with age. Drosophila melanogaster is a useful model to study aging and sleep, and inheriting mutations affecting the potassium current Shaker results in flies that sleep less and have a shorter lifespan. However, whether the link between short sleep and reduced longevity exists also in wild-type flies is unknown. Similarly, it is unknown whether such a link depends on sleep amount per se, rather than on other factors such as waking activity. Also, sleep quality has been shown to decrease in old flies, but it remains unclear whether aging-related sleep fragmentation is a generalized phenomenon. RESULTS: We compared 3 short sleeping mutant lines (Hk1, HkY and Hk2) carrying a mutation in Hyperkinetic, which codes for the beta subunit of the Shaker channel, to wild-type siblings throughout their entire lifespan (all flies kept at 20 degrees C). Hk1 and HkY mutants were short sleeping relative to wild-type controls from day 3 after eclosure, and Hk2 flies became short sleepers about two weeks later. All 3 Hk mutant lines had reduced lifespan relative to wild-type flies. Total sleep time showed a trend to increase in all lines with age, but the effect was most pronounced in Hk1 and HkY flies. In both mutant and wild-type lines sleep quality did not decay with age, but the strong preference for sleep at night declined starting in "middle age". Using Cox regression analysis we found that in Hk1 and HkY mutants and their control lines there was a negative relationship between total sleep amount during the first 2 and 4 weeks of age and hazard (individual risk of death), while no association was found in Hk2 flies and their wild-type controls. Hk1 and HkY mutants and their control lines also showed an association between total daily wake activity over the first 2 and 4 weeks of age and hazard. However, when both sleep duration and wake activity were used in the same regression, the effects of activity were much reduced, while most of the sleep effects remained significant. Finally, Hk1 flies and wild-type siblings were also tested at 25 degrees C, and results were similar to those at 20 degrees C. Namely, Hk1 mutants were short sleeping, hyperactive, and short lived relative to controls, and sleep quality in both groups did not decrease with age. CONCLUSIONS: Different Hk mutations affect the sleep phenotype, and do so in an age-dependent manner. In 4 of the 6 lines tested sleep associates significantly with lifespan variation even after any effect of activity is removed, but activity does not associate significantly with lifespan after the effects of sleep are removed. Thus, in addition to environmental factors and genetic background, sleep may also affect longevity. Sleep quality does not necessarily decay as flies age, suggesting that aging-related sleep fragmentation may also depend on many factors, including genetic background and rearing conditions.
Assuntos
Envelhecimento/genética , Drosophila melanogaster/genética , Longevidade/genética , Mutação/genética , Transtornos do Sono-Vigília/genética , Sono/genética , Animais , Química Encefálica/genética , Quinase 1 do Ponto de Checagem , Proteínas de Drosophila , Predisposição Genética para Doença/genética , Modelos Animais , Fenótipo , Canais de Potássio/genética , Superfamília Shaker de Canais de Potássio/genéticaRESUMO
BACKGROUND: Anesthesia and sleep share physiologic and behavioral similarities. The anesthetic requirement of the recently identified Drosophila mutant minisleeper and other Drosophila mutants was investigated. METHODS: Sleep and wakefulness were determined by measuring activity of individual wild-type and mutant flies. Based on the response of the flies at different concentrations of the volatile anesthetics isoflurane and sevoflurane, concentration-response curves were generated and EC50 values were calculated. RESULTS: The average amount of daily sleep in wild-type Drosophila (n = 64) was 965 +/- 15 min, and 1,022 +/- 29 in Na[har](P > 0.05; n = 32) (mean +/- SEM, all P compared to wild-type and other shaker alleles). Sh flies slept 584 +/- 13 min (n = 64, P < 0.01), Sh flies 412 +/- 22 min (n = 32, P < 0.01), and Sh flies 782 +/- 25 min (n = 32, P < 0.01). The EC50 values for isoflurane were 0.706 (95% CI 0.649 to 0.764, n = 661) and for sevoflurane 1.298 (1.180 to 1.416, n = 522) in wild-type Drosophila; 1.599 (1.527 to 1.671, n = 308) and 2.329 (2.177 to 2.482, n = 282) in Sh, 1.306 (1.212 to 1.400, n = 393) and 2.013 (1.868 to 2.158, n = 550) in Sh, 0.957 (0.860 to 1.054, n = 297) and 1.619 (1.508 to 1.731, n = 386) in Sh, and 0.6154 (0.581 to 0.649, n = 360; P < 0.05) and 0.9339 (0.823 to 1.041, n = 274) in Na[har], respectively (all P < 0.01). CONCLUSIONS: A single-gene mutation in Drosophila that causes an extreme reduction in daily sleep is responsible for a significant increase in the requirement of volatile anesthetics. This suggests that a single gene mutation affects both sleep behavior and anesthesia and sedation.
Assuntos
Anestésicos Inalatórios/farmacologia , Mutação/genética , Mutação/fisiologia , Sono/efeitos dos fármacos , Sono/genética , Alelos , Anestésicos Inalatórios/administração & dosagem , Animais , Relação Dose-Resposta a Droga , Drosophila , Feminino , Isoflurano/administração & dosagem , Isoflurano/farmacologia , Masculino , Atividade Motora/efeitos dos fármacosRESUMO
Animals consolidate some, but not all, learning experiences into long-term memory. Across the animal kingdom, sleep has been found to have a beneficial effect on the consolidation of recently formed memories into long-term storage. However, the underlying mechanisms of sleep dependent memory consolidation are poorly understood. Here, we show that consolidation of courtship long-term memory in Drosophila is mediated by reactivation during sleep of dopaminergic neurons that were earlier involved in memory acquisition. We identify specific fan-shaped body neurons that induce sleep after the learning experience and activate dopaminergic neurons for memory consolidation. Thus, we provide a direct link between sleep, neuronal reactivation of dopaminergic neurons, and memory consolidation.
Assuntos
Corte , Neurônios Dopaminérgicos/fisiologia , Drosophila/fisiologia , Aprendizagem , Consolidação da Memória , Memória de Longo Prazo , Sono , AnimaisRESUMO
Animals employ diverse learning rules and synaptic plasticity dynamics to record temporal and statistical information about the world. However, the molecular mechanisms underlying this diversity are poorly understood. The anatomically defined compartments of the insect mushroom body function as parallel units of associative learning, with different learning rates, memory decay dynamics and flexibility (Aso and Rubin, 2016). Here, we show that nitric oxide (NO) acts as a neurotransmitter in a subset of dopaminergic neurons in Drosophila. NO's effects develop more slowly than those of dopamine and depend on soluble guanylate cyclase in postsynaptic Kenyon cells. NO acts antagonistically to dopamine; it shortens memory retention and facilitates the rapid updating of memories. The interplay of NO and dopamine enables memories stored in local domains along Kenyon cell axons to be specialized for predicting the value of odors based only on recent events. Our results provide key mechanistic insights into how diverse memory dynamics are established in parallel memory systems.
Assuntos
Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Memória/fisiologia , Óxido Nítrico/metabolismo , Óxido Nítrico/farmacologia , Animais , Dopamina/farmacologia , Proteínas de Drosophila , Drosophila melanogaster/fisiologia , Aprendizagem/fisiologia , Corpos Pedunculados/fisiologia , Neurotransmissores/metabolismo , Odorantes , Olfato/fisiologiaRESUMO
In mammals, sleep is thought to be important for health, cognition, and memory. Fruit flies share most features of mammalian sleep, and a recent study found that Drosophila lines carrying loss-of-function mutations in Shaker (Sh) are short sleeping, suggesting that the Sh current plays a major role in regulating daily sleep amount. The Sh current is potentiated by a beta modulatory subunit coded by Hyperkinetic (Hk). Here, we demonstrate that severe loss-of-function mutations of Hk reduce sleep and do so primarily by affecting the Sh current. Moreover, we prove, using a transgenic approach, that a wild-type copy of Hk is sufficient to restore normal sleep. Furthermore, we show that short-sleeping Hk mutant lines have a memory deficit, whereas flies carrying a weaker hypomorphic Hk allele have normal sleep and normal memory. By comparing six short-sleeping Sh lines with two normal sleeping ones, we also found that only alleles that reduce sleep also impair memory. These data identify a gene, Hk, which is necessary to maintain normal sleep, and provide genetic evidence that short sleep and poor memory are linked.
Assuntos
Proteínas de Drosophila/genética , Transtornos da Memória/genética , Mutação , Canais de Potássio/genética , Sono/genética , Animais , Animais Geneticamente Modificados , Drosophila , Proteínas de Drosophila/metabolismo , Hipercinese/genética , Hipercinese/metabolismo , Transtornos da Memória/metabolismo , Canais de Potássio/fisiologia , Superfamília Shaker de Canais de Potássio/genéticaRESUMO
Sleep is present and tightly regulated in every vertebrate species in which it has been carefully investigated, but what sleep is for remains a mystery. Sleep is also present in invertebrates, and an extensive analysis in Drosophila melanogaster has shown that sleep in fruit flies shows most of the fundamental features that characterize sleep in mammals. In Drosophila, sleep consists of sustained periods of quiescence associated with an increased arousal threshold. Fly sleep is modulated by several of the same stimulants and hypnotics that affect mammalian sleep. Moreover, like in mammals, fly sleep shows remarkable interindividual variability. The expression of several genes involved in energy metabolism, synaptic plasticity, and the response to cellular stress varies in Drosophila between sleep and wakefulness, and the same occurs in rodents. Brain activity also changes in flies as a function of behavioral state. Furthermore, Drosophila sleep is tightly regulated in a circadian and homeostatic manner, and the homeostatic regulation is largely independent of the circadian regulation. After sleep deprivation, recovery sleep in flies is longer in duration and more consolidated, indicated by an increase in arousal threshold and fewer brief awakenings. Finally, sleep deprivation in flies impairs vigilance and performance. Because of the extensive similarities between flies and mammals, Drosophila is now being used as a promising model system for the genetic dissection of sleep. Over the last few years, mutagenesis screens have isolated several short sleeping mutants, a demonstration that single genes can have a powerful effect on a complex trait like sleep.
Assuntos
Drosophila melanogaster/fisiologia , Sono/fisiologia , Vigília/fisiologia , Animais , Drosophila melanogaster/genética , Mutação/genética , Sono/genética , Vigília/genéticaRESUMO
We have developed a series of yellow genetically encoded Ca2+ indicators for optical imaging (Y-GECOs) with inverted responses to Ca2+ and apparent dissociation constants (Kd') ranging from 25 to 2400 nM. To demonstrate the utility of this affinity series of Ca2+ indicators, we expressed the four highest affinity variants (Kd's = 25, 63, 121, and 190 nM) in the Drosophila medulla intrinsic neuron Mi1. Hyperpolarization of Mi1 by optogenetic stimulation of the laminar monopolar neuron L1 produced a decrease in intracellular Ca2+ in layers 8-10, and a corresponding increase in Y-GECO fluorescence. These experiments revealed that lower Kd' was associated with greater increases in fluorescence, but longer delays to reach the maximum signal change due to slower off-rate kinetics.
Assuntos
Cálcio/metabolismo , Optogenética/métodos , Animais , Sinalização do Cálcio/fisiologia , Drosophila , Cinética , Neurônios/metabolismo , Imagem Óptica/métodosRESUMO
Exploration of a novel environment leads to neuronal DNA double-strand breaks (DSBs). These DSBs are generated by type 2 topoisomerase to relieve topological constrains that limit transcription of plasticity-related immediate early genes. If not promptly repaired, however, DSBs may lead to cell death. Since the induction of plasticity-related genes is higher in wake than in sleep, we asked whether it is specifically wake associated with synaptic plasticity that leads to DSBs, and whether sleep provides any selective advantage over wake in their repair. In flies and mice, we find that enriched wake, more than simply time spent awake, induces DSBs, and their repair in mice is delayed or prevented by subsequent wake. In both species the repair of irradiation-induced neuronal DSBs is also quicker during sleep, and mouse genes mediating the response to DNA damage are upregulated in sleep. Thus, sleep facilitates the repair of neuronal DSBs.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Neurônios/fisiologia , Sono , Animais , Encéfalo/patologia , Encéfalo/efeitos da radiação , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Drosophila melanogaster , Feminino , Masculino , Camundongos Endogâmicos C57BL , Neurônios/efeitos da radiação , Lesões Experimentais por Radiação/genética , Ativação TranscricionalRESUMO
In Drosophila melanogaster, the w(+) transgene in P[lacW]ci(Dplac) is uniformly expressed throughout the adult eye. However, when other P elements are present, this w(+) transgene is randomly silenced and this produces a variegated eye phenotype. This P-element-dependent silencing (PDS) is limited to w(+) transgenes inserted in a specific region on chromosome 4. In a screen for genetic modifiers of PDS, we isolated mutations in Su(var)205, Su(var)3-7, and two unidentified genes that suppress this variegated phenotype. Therefore, only a few of the genes encoding heterochromatic modifiers act dose dependently in PDS. In addition, we recovered two spontaneous mutations of P[lacW]ci(Dplac) that variegate in the absence of P elements. These P[lacW]i(Dplac) derivatives have a gypsy element inserted proximally to the P[lacW]ci(Dplac) insert. The same mutations that suppress PDS also suppress w(+) silencing from these P[lacW]ci(Dplac) derivative alleles. This indicates that both cis-acting changes in sequence and trans-acting P elements cause a similar change in chromatin structure that silences w(+) expression in P[lacW]ci(Dplac). Together, these results confirm that PDS occurs at P[lacW]ci(Dplac) because of the chromatin structure at this chromosomal position. Studying w(+) variegation from P[lacW]ci(Dplac) provides a model for the interactions that can enhance heterochromatic silencing at single P-element inserts.
Assuntos
Proteínas Cromossômicas não Histona/genética , Elementos de DNA Transponíveis , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Regulação da Expressão Gênica/fisiologia , Inativação Gênica , Animais , Animais Geneticamente Modificados , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Olho/metabolismo , Dosagem de Genes , Genes Dominantes , Heterocromatina/genética , Pigmentação/genéticaRESUMO
Sleep consists of quiescent periods with reduced responsiveness to external stimuli. Despite being maladaptive in that when asleep, animals are less able to respond to dangerous stimuli; sleep behavior is conserved in all animal species studied to date. Thus, sleep must be performing at least one fundamental, conserved function that is necessary, and/or whose benefits outweigh its maladaptive consequences. Currently, there is no consensus on what that function might be. Over the last 10 years, multiple groups have started to characterize the molecular mechanisms and brain structures necessary for normal sleep in Drosophila melanogaster. These researchers are exploiting genetic tools developed in Drosophila over the past century to identify and manipulate gene expression. Forward genetic screens can identify molecular components in complex biological systems and once identified, these genes can be manipulated within specific brain areas to determine which neuronal groups are important to initiate and maintain sleep. Screening for mutations and brain regions necessary for normal sleep has revealed that several genes that affect sleep are involved in synaptic plasticity and have preferential expression in the mushroom bodies (MBs). Moreover, altering MB neuronal activity alters sleep. Previous genetic screens found that the same genes enriched in MB are necessary for learning and memory. Increasing evidence in mammals, including humans, points to a beneficial role for sleep in synaptic plasticity, learning and memory. Thus, results from both flies and mammals suggest a strong link between sleep need and wake plasticity.