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1.
Proc Natl Acad Sci U S A ; 119(50): e2211308119, 2022 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-36469774

RESUMO

Learned experiences are not necessarily consolidated into long-term memory (LTM) unless they are periodic and meaningful. LTM depends on de novo protein synthesis mediated by cyclic AMP response element-binding protein (CREB) activity. In Drosophila, two creb genes (crebA, crebB) and multiple CREB isoforms have reported influences on aversive olfactory LTM in response to multiple cycles of spaced conditioning. How CREB isoforms regulate LTM effector genes in various neural elements of the memory circuit is unclear, especially in the mushroom body (MB), a prominent associative center in the fly brain that has been shown to participate in LTM formation. Here, we report that i) spaced training induces crebB expression in MB α-lobe neurons and ii) elevating specific CREBB isoform levels in the early α/ß subpopulation of MB neurons enhances LTM formation. By contrast, learning from weak training iii) induces 5-HT1A serotonin receptor synthesis, iv) activates 5-HT1A in early α/ß neurons, and v) inhibits LTM formation. vi) LTM is enhanced when this inhibitory effect is relieved by down-regulating 5-HT1A or overexpressing CREBB. Our findings show that spaced training-induced CREBB antagonizes learning-induced 5-HT1A in early α/ß MB neurons to modulate LTM consolidation.


Assuntos
Proteínas de Drosophila , Corpos Pedunculados , Animais , Corpos Pedunculados/fisiologia , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Memória de Longo Prazo/fisiologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Drosophila melanogaster/metabolismo
2.
Proc Natl Acad Sci U S A ; 118(37)2021 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-34507985

RESUMO

Episodic events are frequently consolidated into labile memory but are not necessarily transferred to persistent long-term memory (LTM). Regulatory mechanisms leading to LTM formation are poorly understood, however, especially at the resolution of identified neurons. Here, we demonstrate enhanced LTM following aversive olfactory conditioning in Drosophila when the transcription factor cyclic AMP response element binding protein A (CREBA) is induced in just two dorsal-anterior-lateral (DAL) neurons. Our experiments show that this process is regulated by protein-gene interactions in DAL neurons: (1) crebA transcription is induced by training and repressed by crebB overexpression, (2) CREBA bidirectionally modulates LTM formation, (3) crebA overexpression enhances training-induced gene transcription, and (4) increasing membrane excitability enhances LTM formation and gene expression. These findings suggest that activity-dependent gene expression in DAL neurons during LTM formation is regulated by CREB proteins.


Assuntos
Proteína A de Ligação a Elemento de Resposta do AMP Cíclico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas de Drosophila/metabolismo , Memória de Longo Prazo/fisiologia , Transativadores/metabolismo , Animais , Condicionamento Clássico/fisiologia , Condicionamento Psicológico , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Proteína A de Ligação a Elemento de Resposta do AMP Cíclico/genética , Proteína A de Ligação a Elemento de Resposta do AMP Cíclico/fisiologia , Proteínas de Drosophila/fisiologia , Drosophila melanogaster , Feminino , Expressão Gênica/genética , Regulação da Expressão Gênica/genética , Masculino , Neurônios/metabolismo , Neurônios/fisiologia , Percepção Olfatória/fisiologia , Olfato/fisiologia , Transativadores/fisiologia
3.
J Neurogenet ; 35(3): 112-116, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34256677

RESUMO

A comprehensive science, technology, engineering, and mathematics (STEM) education has persistent formative effects on individuals, communities, and society. In this regard, Marla Sokolowski's academic legacy will forever reflect her unique contributions to STEM education and mentoring. Furthermore, her creative and multidisciplinary approach to research has resulted in groundbreaking advances in our understanding of behavior genetics. Illustrated here are a few of our life-long learning experiences drawn mainly from earlier parts of Marla's career.


Assuntos
Genética/história , História do Século XX , História do Século XXI
4.
J Neurogenet ; 34(1): 69-82, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31965871

RESUMO

Neuronal development and memory consolidation are conserved processes that rely on nuclear-cytoplasmic transport of signaling molecules to regulate gene activity and initiate cascades of downstream cellular events. Surprisingly, few reports address and validate this widely accepted perspective. Here we show that Importin-α2 (Imp-α2), a soluble nuclear transporter that shuttles cargoes between the cytoplasm and nucleus, is vital for brain development, learning and persistent memory in Drosophila melanogaster. Mutations in importin-α2 (imp-α2, known as Pendulin or Pen and homologous with human KPNA2) are alleles of mushroom body miniature B (mbmB), a gene known to regulate aspects of brain development and influence adult behavior in flies. Mushroom bodies (MBs), paired associative centers in the brain, are smaller than normal due to defective proliferation of specific intrinsic Kenyon cell (KC) neurons in mbmB mutants. Extant KCs projecting to the MB ß-lobe terminate abnormally on the contralateral side of the brain. mbmB adults have impaired olfactory learning but normal memory decay in most respects, except that protein synthesis-dependent long-term memory (LTM) is abolished. This observation supports an alternative mechanism of persistent memory in which mutually exclusive protein-synthesis-dependent and -independent forms rely on opposing cellular mechanisms or circuits. We propose a testable model of Imp-α2 and nuclear transport roles in brain development and conditioned behavior. Based on our molecular characterization, we suggest that mbmB is hereafter referred to as imp-α2mbmB.


Assuntos
Encéfalo/fisiologia , Aprendizagem/fisiologia , Consolidação da Memória/fisiologia , Neurogênese/fisiologia , alfa Carioferinas/metabolismo , Animais , Animais Geneticamente Modificados , Encéfalo/embriologia , Drosophila melanogaster , alfa Carioferinas/genética
5.
J Exp Biol ; 221(Pt 1)2018 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-29061687

RESUMO

Brain development and behavior are sensitive to a variety of environmental influences including social interactions and physicochemical stressors. Sensory input in situ is a mosaic of both enrichment and stress, yet little is known about how multiple environmental factors interact to affect brain anatomical structures, circuits and cognitive function. In this study, we addressed these issues by testing the individual and combined effects of sub-adult thermal stress, larval density and early-adult living spatial enrichment on brain anatomy and olfactory associative learning in adult Drosophila melanogaster In response to heat stress, the mushroom bodies (MBs) were the most volumetrically impaired among all of the brain structures, an effect highly correlated with reduced odor learning performance. However, MBs were not sensitive to either larval culture density or early-adult living conditions. Extreme larval crowding reduced the volume of the antennal lobes, optic lobes and central complex. Neither larval crowding nor early-adult spatial enrichment affected olfactory learning. These results illustrate that various brain structures react differently to environmental inputs, and that MB development and learning are highly sensitive to certain stressors (pre-adult hyperthermia) and resistant to others (larval crowding).


Assuntos
Drosophila melanogaster/anatomia & histologia , Drosophila melanogaster/fisiologia , Meio Ambiente , Temperatura Alta/efeitos adversos , Percepção Olfatória , Olfato , Animais , Encéfalo/anatomia & histologia , Encéfalo/crescimento & desenvolvimento , Drosophila melanogaster/crescimento & desenvolvimento , Larva/anatomia & histologia , Larva/crescimento & desenvolvimento , Larva/fisiologia , Aprendizagem , Densidade Demográfica
6.
Curr Biol ; 34(5): 946-957.e4, 2024 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-38320552

RESUMO

Animals have complementary parallel memory systems that process signals from various sensory modalities. In the brain of the fruit fly Drosophila melanogaster, mushroom body (MB) circuitry is the primary associative neuropil, critical for all stages of olfactory memory. Here, our findings suggest that active signaling from specific asymmetric body (AB) neurons is also crucial for this process. These AB neurons respond to odors and electric shock separately and exhibit timing-sensitive neuronal activity in response to paired stimulation while leaving a decreased memory trace during retrieval. Our experiments also show that rutabaga-encoded adenylate cyclase, which mediates coincidence detection, is required for learning and short-term memory in both AB and MB. We observed additive effects when manipulating rutabaga co-expression in both structures. Together, these results implicate the AB in playing a critical role in associative olfactory learning and short-term memory.


Assuntos
Proteínas de Drosophila , Drosophila , Animais , Drosophila/metabolismo , Drosophila melanogaster/fisiologia , Neurônios/fisiologia , Aprendizagem/fisiologia , Encéfalo/metabolismo , Proteínas de Drosophila/metabolismo , Olfato/fisiologia , Corpos Pedunculados/fisiologia
7.
Cell Rep ; 42(8): 112974, 2023 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-37590142

RESUMO

Long-term memory (LTM) requires learning-induced synthesis of new proteins allocated to specific neurons and synapses in a neural circuit. Not all learned information, however, becomes permanent memory. How the brain gates relevant information into LTM remains unclear. In Drosophila adults, weak learning after a single training session in an olfactory aversive task typically does not induce protein-synthesis-dependent LTM. Instead, strong learning after multiple spaced training sessions is required. Here, we report that pre-synaptic active-zone protein synthesis and cholinergic signaling from the early α/ß subset of mushroom body (MB) neurons produce a downstream inhibitory effect on LTM formation. When we eliminated inhibitory signaling from these neurons, weak learning was then sufficient to form LTM. This bidirectional circuit mechanism modulates the transition between distinct memory phase functions in different subpopulations of MB neurons in the olfactory memory circuit.

8.
Learn Mem ; 18(4): 250-3, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21441302

RESUMO

Associative conditioning in Drosophila melanogaster has been well documented for several decades. However, most studies report only simple associations of conditioned stimuli (CS, e.g., odor) with unconditioned stimuli (US, e.g., electric shock) to measure learning or establish memory. Here we describe a straightforward second-order conditioning (SOC) protocol that further demonstrates the flexibility of fly behavior. In SOC, a previously conditioned stimulus (CS1) is used as reinforcement for a second conditioned stimulus (CS2) in associative learning. This higher-order context presents an opportunity for reassessing the roles of known learning and memory genes and neuronal networks in a new behavioral paradigm.


Assuntos
Condicionamento Clássico/fisiologia , Drosophila/fisiologia , Olfato/fisiologia , Análise de Variância , Animais , Comportamento Animal , Aprendizagem por Discriminação/fisiologia , Estimulação Elétrica/efeitos adversos , Extinção Psicológica , Aprendizagem em Labirinto/fisiologia , Odorantes , Reforço Psicológico
9.
iScience ; 24(12): 103506, 2021 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-34934925

RESUMO

Long-term memory (LTM) formation requires consolidation processes to overcome interfering signals that erode memory formation. Olfactory memory in Drosophila involves convergent projection neuron (PN; odor) and dopaminergic neuron (DAN; reinforcement) input to the mushroom body (MB). How post-training DAN activity in the posterior lateral protocerebrum (PPL1) continues to regulate memory consolidation remains unknown. Here we address this question using targeted transgenes in behavior and electrophysiology experiments to show that (1) persistent post-training activity of PPL1-α2α'2 and PPL1-α3 DANs interferes with aversive LTM formation; (2) neuropeptide F (NPF) signaling blocks this interference in PPL1-α2α'2 and PPL1-α3 DANs after spaced training to enable LTM formation; and (3) training-induced NPF release and neurotransmission from two upstream dorsal-anterior-lateral (DAL2) neurons are required to form LTM. Thus, NPF signals from DAL2 neurons to specific PPL1 DANs disinhibit the memory circuit, ensuring that periodic events are remembered as consolidated LTM.

10.
J Neurogenet ; 23(1-2): 173-84, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19145515

RESUMO

The central body (or central complex, CCX) and the mushroom bodies (MBs) are brain structures in most insect phyla that have been shown to influence aspects of locomotion. The CCX regulates motor coordination and enhances activity while MBs have, thus far, been shown to suppress motor activity levels measured over time intervals ranging from hours to weeks. In this report, we investigate MB involvement in motor behavior during the initial stages (15 minutes) of walking in Buridan's paradigm. We measured aspects of walking in flies that had MB lesions induced by mutations in six different genes and by chemical ablation. All tested flies were later examined histologically to assess MB neuroanatomy. Mutant strains with MB structural defects were generally less active in walking than wild-type flies. Most mutants in which MBs were also ablated with hydroxyurea (HU) showed additional activity decrements. Variation in measures of velocity and orientation to landmarks among wild-type and mutant flies was attributed to pleiotropy, rather than to MB lesions. We conclude that MBs upregulate activity during the initial stages of walking, but suppress activity thereafter. An MB influence on decision making has been shown in a wide range of complex behaviors. We suggest that MBs provide appropriate contextual information to motor output systems in the brain, indirectly fine tuning walking by modifying the quantity (i.e., activity) of behavior.


Assuntos
Drosophila/fisiologia , Atividade Motora/fisiologia , Corpos Pedunculados/fisiologia , Animais , Encéfalo/patologia , Encéfalo/fisiologia , Feminino , Hidroxiureia , Masculino , Corpos Pedunculados/patologia , Mutação , Orientação/fisiologia , Fenótipo , Estimulação Luminosa/métodos
11.
Brain Res ; 1712: 158-166, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30711401

RESUMO

The Drosophila olfactory system provides an excellent model to elucidate the neural circuits that control behaviors elicited by environmental stimuli. Despite significant progress in defining olfactory circuit components and their connectivity, little is known about the mechanisms that transfer the information from the primary antennal olfactory receptor neurons to the higher order brain centers. Here, we show that the Dystrophin Dp186 isoform is required in the olfactory system circuit for olfactory functions. Using two-photon calcium imaging, we found the reduction of calcium influx in olfactory receptor neurons (ORNs) and also the defect of GABAA mediated inhibitory input in the projection neurons (PNs) in Dp186 mutation. Moreover, the Dp186 mutant flies which display a decreased odor avoidance behavior were rescued by Dp186 restoration in the Drosophila olfactory neurons in either the presynaptic ORNs or the postsynaptic PNs. Therefore, these results revealed a role for Dystrophin, Dp 186 isoform in gain control of the olfactory synapse via the modulation of excitatory and inhibitory synaptic inputs to olfactory projection neurons.


Assuntos
Distrofina/metabolismo , Condutos Olfatórios/fisiologia , Olfato/fisiologia , Animais , Cálcio/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Distrofina/fisiologia , Feminino , Interneurônios/metabolismo , Masculino , Odorantes , Percepção Olfatória/fisiologia , Neurônios Receptores Olfatórios/fisiologia , Sinapses/fisiologia
13.
Aging (Albany NY) ; 8(5): 1115-34, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-27025190

RESUMO

Accumulating evidence suggests that early-life diet may program one's health status by causing permanent alternations in specific organs, tissues, or metabolic or homeostatic pathways, and such programming effects may propagate across generations through heritable epigenetic modifications. However, it remains uninvestigated whether postnatal dietary changes may program longevity across generations. To address this question of important biological and public health implications, newly-born flies (F0) were collected and subjected to various post-eclosion dietary manipulations (PDMs) with different protein-carbohydrate (i.e., LP, IP or HP for low-, intermediate- or high-protein) contents or a control diet (CD). Longevity and fecundity analyses were performed with these treated F0 flies and their F1, F2 and F3 offspring, while maintained on CD at all times. The LP and HP PDMs shortened longevity, while the IP PDM extended longevity significantly up to the F3 generation. Furthermore, the LP reduced while the IP PDM increased lifetime fecundity across the F0-F2 generations. Our observations establish the first animal model for studying transgenerational inheritance of nutritional programming of longevity, making it possible to investigate the underlying epigenetic mechanisms and identify gene targets for drug discovery in future studies.


Assuntos
Dieta , Epigênese Genética/fisiologia , Longevidade/fisiologia , Animais , Carboidratos da Dieta , Proteínas Alimentares , Drosophila melanogaster , Feminino , Masculino , Reprodução/fisiologia
14.
Cold Spring Harb Protoc ; 2012(2): 235-8, 2012 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-22301645

RESUMO

The Drosophila nervous system provides a valuable model for studying various aspects of brain development and function. The postembryonic Drosophila brain is especially useful, because specific neuron types derive from specific progenitors at specific times. Elucidating the means by which diverse neuron types derive from a limited number of progenitors can contribute significantly to our understanding of the genetic and molecular mechanisms involved in developmental neurobiology. Antibody-labeling techniques are particularly useful for examining the Drosophila brain. These methods generally use primary antibodies specific to a protein or a structure of interest and a fluorescently labeled or enzyme-coupled secondary antibody to detect the primary antibodies. Immunofluorescence methods allow for simultaneous probing for multiple antigens using different fluorophores, as well as high-resolution confocal examination of deep structures. This protocol describes general procedures for antibody labeling of neural tissue from Drosophila, as well as visualization techniques for fluorescent and enzyme-linked probes.


Assuntos
Anticorpos , Sistema Nervoso Central/citologia , Sistema Nervoso Central/crescimento & desenvolvimento , Biologia do Desenvolvimento/métodos , Drosophila/citologia , Drosophila/crescimento & desenvolvimento , Coloração e Rotulagem/métodos , Animais , Imunofluorescência/métodos , Microscopia Confocal
15.
Cold Spring Harb Protoc ; 2012(2): 239-41, 2012 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-22301646

RESUMO

The Drosophila nervous system provides a valuable model for studying various aspects of brain development and function. The postembryonic Drosophila brain is especially useful, because specific neuron types derive from specific progenitors at specific times. Elucidating the means by which diverse neuron types derive from a limited number of progenitors can contribute significantly to our understanding of the genetic and molecular mechanisms involved in developmental neurobiology. ß-Galactosidase, the product of the E. coli lacZ gene, has been used extensively as a reporter in Drosophila research. Staining for ß-galactosidase activity can be performed using the substrate X-gal (5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside), which produces a blue precipitate visible by light microscopy. This detection method is highly sensitive and has the advantage that the results can be observed without the need for specialized microscopy equipment. This protocol describes general procedures for X-gal labeling of neural tissue from Drosophila.


Assuntos
Sistema Nervoso Central/citologia , Sistema Nervoso Central/crescimento & desenvolvimento , Biologia do Desenvolvimento/métodos , Drosophila/citologia , Drosophila/crescimento & desenvolvimento , Galactosídeos , Indóis , Coloração e Rotulagem/métodos , Animais , Genes Reporter , Histocitoquímica/métodos , beta-Galactosidase/genética , beta-Galactosidase/metabolismo
16.
Cold Spring Harb Protoc ; 2012(2): 231-4, 2012 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-22301647

RESUMO

Chemical ablation is an effective tool for studying nervous system development and function in Drosophila. Hydroxyurea (HU) inhibits ribonucleotide reductase, blocking DNA synthesis, and killing dividing cells. The specificity of HU ablation is thus dependent on developmental events. In this respect, HU is useful in determining temporal patterns of neuroblast proliferation and the origins of neuronal elements in flies and other insects. In Drosophila, an especially fortuitous time window occurs at the end of embryonic development. For the first 8-12 h after larval hatching, only five neuroblasts are proliferating in each brain hemisphere. Four of these are found in the dorsal protocerebrum and give rise to the intrinsic elements (Kenyon cells [KCs] and glia) of the mushroom bodies (MBs). The remaining single neuroblast has an anterolateral position in the brain and is the progenitor of local interneurons (LocI) in the antennal lobe (AL) and a subset of lateral relay interneurons (RIl) in the inner antennocerebral tract (iACT). Treating newly hatched larvae with HU results in adult flies with KCs and AL interneurons of embryonic origin only. This protocol describes methods for collecting newly hatched Drosophila larvae and treating them with HU.


Assuntos
Técnicas de Ablação/métodos , Drosophila/embriologia , Drosophila/fisiologia , Hidroxiureia/metabolismo , Corpos Pedunculados/fisiologia , Animais , Drosophila/efeitos dos fármacos , Hidroxiureia/toxicidade , Larva/efeitos dos fármacos , Larva/fisiologia , Corpos Pedunculados/embriologia
17.
Cold Spring Harb Protoc ; 2012(6): 726-32, 2012 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-22661442

RESUMO

The selective removal of cells by ablation is a powerful tool in the study of eukaryotic developmental biology, providing much information about their origin, fate, or function in the developing organism. In Drosophila, three main methods have been used to ablate cells: chemical, genetic, and laser ablation. Each method has its own applicability with regard to developmental stage and the cells to be ablated, and its own limitations. The primary advantage of laser-based ablation is the flexibility provided by the method: The operations can be performed in any cell pattern and at any time in development. Laser-based techniques permit manipulation of structures within cells, even to the molecular level. They can also be used for gene activation. However, laser ablation can be expensive, labor-intensive, and time-consuming. Although live cells can be difficult to image in Drosophila embryos, the use of vital fluorescent imaging methods has made laser-mediated cell manipulation methods more appealing; the methods are relatively straightforward. This article provides the information necessary for setting up and using a laser microscope for lasesr ablation studies.


Assuntos
Técnicas de Ablação/métodos , Drosophila/embriologia , Entomologia/métodos , Lasers , Microscopia/métodos , Animais
18.
Cold Spring Harb Protoc ; 2012(6): 691-3, 2012 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-22661443

RESUMO

Cell ablation is a powerful tool in the study of eukaryotic developmental biology. The selective removal of cells by ablation may provide much information about their origin, fate, or function in the developing organism. Laser-based techniques have an advantage over genetic or chemical ablation methods in that the operations can be performed in essentially any cell pattern and at any time in development. This protocol describes the methods needed to target and ablate specific cells of interest in Drosophila embryos with lasers.


Assuntos
Técnicas de Ablação/métodos , Drosophila/embriologia , Entomologia/métodos , Lasers , Animais
19.
Cold Spring Harb Protoc ; 2012(9): 950-6, 2012 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-22949708

RESUMO

The selective removal of cells by ablation is a powerful tool in the study of eukaryotic developmental biology, providing much information about the origin, fate, or function of these cells in the developing organism. In Drosophila, three main methods have been used to ablate cells: chemical, genetic, and laser ablation. Each method has its own applicability with regard to developmental stage and the cells to be ablated, and its own limitations. This article describes genetic systems for functional cell ablation in Drosophila. Genetic ablation consists of delivering a toxin or death-inducing gene under the control of a cell-specific enhancer, or by means of the GAL4 system. Because of the wide range of existing enhancers, toxins and death genes can be targeted to virtually any cell of choice, allowing for cell-type-specificity. Genetic ablation is less expensive and less labor-intensive than laser ablation. It allows one to analyze the effects of eliminating every cell of a given type within an embryo, and also allows the examination of populations rather than individuals.


Assuntos
Biologia do Desenvolvimento/métodos , Drosophila/citologia , Drosophila/genética , Entomologia/métodos , Técnicas Genéticas , Técnicas de Ablação/métodos , Animais
20.
Cold Spring Harb Protoc ; 2012(9): 1013-6, 2012 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-22949709

RESUMO

The terminal deoxynucleotide transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) method for monitoring targeted cell ablation is based on the in situ labeling of DNA fragmentation sites in nuclei of intact fixed cells. Unlike other methods of detecting dying cells, the use of fixed material allows antigen expression to be monitored at the same time that apoptosis is confirmed in the targeted cells. Double-labeling of Drosophila embryos using the TUNEL reaction and fluorescently tagged antibodies can be adapted to the selected antigen. For some antigens, it is preferable that the TUNEL reaction be performed first, whereas for others, the TUNEL reaction should follow antigen detection. This may be because some antigens may not survive the 37°C incubation or the conditions of the reaction. Similarly, increased fixation times yield better results for some antigens, but not for others. This protocol describes a TUNEL reaction adapted for use on Drosophila embryos in conjunction with fluorescently labeled antibodies.


Assuntos
Técnicas de Ablação/métodos , Anticorpos/metabolismo , Drosophila/embriologia , Entomologia/métodos , Marcação In Situ das Extremidades Cortadas/métodos , Microscopia de Fluorescência/métodos , Coloração e Rotulagem/métodos , Animais
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