RESUMEN
The isocortex and hippocampal formation (HPF) in the mammalian brain play critical roles in perception, cognition, emotion, and learning. We profiled â¼1.3 million cells covering the entire adult mouse isocortex and HPF and derived a transcriptomic cell-type taxonomy revealing a comprehensive repertoire of glutamatergic and GABAergic neuron types. Contrary to the traditional view of HPF as having a simpler cellular organization, we discover a complete set of glutamatergic types in HPF homologous to all major subclasses found in the six-layered isocortex, suggesting that HPF and the isocortex share a common circuit organization. We also identify large-scale continuous and graded variations of cell types along isocortical depth, across the isocortical sheet, and in multiple dimensions in hippocampus and subiculum. Overall, our study establishes a molecular architecture of the mammalian isocortex and hippocampal formation and begins to shed light on its underlying relationship with the development, evolution, connectivity, and function of these two brain structures.
Asunto(s)
Hipocampo/citología , Neocórtex/citología , Transcriptoma/genética , Animales , Neuronas GABAérgicas/citología , Neuronas GABAérgicas/metabolismo , Ácido Glutámico/metabolismo , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
Gene targeting is an incredibly valuable technique. Sometimes, however, it can also be extremely challenging for various intrinsic reasons (e.g. low target accessibility or nature/extent of gene modification). To bypass these barriers, we designed a transgene-based system in Drosophila that increases the number of independent gene targeting events while at the same time enriching for correctly targeted progeny. Unfortunately, with particularly challenging gene targeting experiments, our original design yielded numerous false positives. Here, we deliver a much-improved technique, named Enhanced Golic+ (E-Golic+). E-Golic+ incorporates genetic modifications to tighten lethality-based selection while simultaneously boosting efficiency. With E-Golic+, we easily achieve previously unattainable gene targeting. Additionally, we built an E-Golic+-based, high-efficiency genetic pipeline for transgene swapping. We demonstrate its utility by transforming GAL4 enhancer-trap lines into tissue-specific Cas9-expressing lines. Given the superior efficiency, specificity and scalability, E-Golic+ promises to expedite development of additional sophisticated genetic/genomic tools in Drosophila.
Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Drosophila/metabolismo , Marcación de Gen/métodos , Transgenes/genética , Animales , Animales Modificados Genéticamente/genética , Animales Modificados Genéticamente/metabolismo , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Femenino , Células Germinativas/citología , Células Germinativas/metabolismo , Masculino , Regiones Promotoras Genéticas , ARN Guía de Kinetoplastida/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Macroglial cells in the central nervous system exhibit regional specialization and carry out region-specific functions. Diverse glial cells arise from specific progenitors in specific spatiotemporal patterns. This raises an interesting possibility that glial precursors with distinct developmental fates exist that govern region-specific gliogenesis. Here, we have mapped the glial progeny produced by the Drosophila type II neuroblasts, which, like vertebrate radial glia cells, yield both neurons and glia via intermediate neural progenitors (INPs). Distinct type II neuroblasts produce different characteristic sets of glia. A single INP can make both astrocyte-like and ensheathing glia, which co-occupy a relatively restrictive subdomain. Blocking apoptosis uncovers further lineage distinctions in the specification, proliferation and survival of glial precursors. Both the switch from neurogenesis to gliogenesis and the subsequent glial expansion depend on Notch signaling. Taken together, lineage origins preconfigure the development of individual glial precursors with involvement of serial Notch actions in promoting gliogenesis.
Asunto(s)
Encéfalo/embriología , Proteínas de Drosophila/metabolismo , Drosophila/embriología , Células-Madre Neurales/citología , Neurogénesis/fisiología , Receptores Notch/metabolismo , Animales , Apoptosis/fisiología , Astrocitos/citología , Encéfalo/citología , Linaje de la Célula/fisiología , Proliferación Celular/fisiología , Neuronas/citologíaRESUMEN
In their classic experiments, Olds and Milner showed that rats learn to lever press to receive an electric stimulus in specific brain regions. This led to the identification of mammalian reward centers. Our interest in defining the neuronal substrates of reward perception in the fruit fly Drosophila melanogaster prompted us to develop a simpler experimental approach wherein flies could implement behavior that induces self-stimulation of specific neurons in their brains. The high-throughput assay employs optogenetic activation of neurons when the fly occupies a specific area of a behavioral chamber, and the flies' preferential occupation of this area reflects their choosing to experience optogenetic stimulation. Flies in which neuropeptide F (NPF) neurons are activated display preference for the illuminated side of the chamber. We show that optogenetic activation of NPF neuron is rewarding in olfactory conditioning experiments and that the preference for NPF neuron activation is dependent on NPF signaling. Finally, we identify a small subset of NPF-expressing neurons located in the dorsomedial posterior brain that are sufficient to elicit preference in our assay. This assay provides the means for carrying out unbiased screens to map reward neurons in flies.
Asunto(s)
Proteínas de Drosophila/metabolismo , Neuronas/metabolismo , Neuropéptidos/metabolismo , Transducción de Señal/fisiología , Animales , Proteínas de Drosophila/genética , Drosophila melanogaster , Neuropéptidos/genéticaRESUMEN
A brain consists of numerous distinct neurons arising from a limited number of progenitors, called neuroblasts in Drosophila. Each neuroblast produces a specific neuronal lineage. To unravel the transcriptional networks that underlie the development of distinct neuroblast lineages, we marked and isolated lineage-specific neuroblasts for RNA sequencing. We labeled particular neuroblasts throughout neurogenesis by activating a conditional neuroblast driver in specific lineages using various intersection strategies. The targeted neuroblasts were efficiently recovered using a custom-built device for robotic single-cell picking. Transcriptome analysis of mushroom body, antennal lobe and type II neuroblasts compared with non-selective neuroblasts, neurons and glia revealed a rich repertoire of transcription factors expressed among neuroblasts in diverse patterns. Besides transcription factors that are likely to be pan-neuroblast, many transcription factors exist that are selectively enriched or repressed in certain neuroblasts. The unique combinations of transcription factors present in different neuroblasts may govern the diverse lineage-specific neuron fates.
Asunto(s)
Linaje de la Célula/genética , Drosophila melanogaster/genética , Marcación de Gen , Neuronas/citología , Robótica , Transcriptoma/genética , Animales , Animales Modificados Genéticamente , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Regulación del Desarrollo de la Expresión Génica , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Factores de Transcripción/metabolismoRESUMEN
Drosophila type II neuroblasts (NBs), like mammalian neural stem cells, deposit neurons through intermediate neural progenitors (INPs) that can each produce a series of neurons. Both type II NBs and INPs exhibit age-dependent expression of various transcription factors, potentially specifying an array of diverse neurons by combinatorial temporal patterning. Not knowing which mature neurons are made by specific INPs, however, conceals the actual variety of neuron types and limits further molecular studies. Here we mapped neurons derived from specific type II NB lineages and found that sibling INPs produced a morphologically similar but temporally regulated series of distinct neuron types. This suggests a common fate diversification program operating within each INP that is modulated by NB age to generate slightly different sets of diverse neurons based on the INP birth order. Analogous mechanisms might underlie the expansion of neuron diversity via INPs in mammalian brain.
Asunto(s)
Drosophila/citología , Drosophila/crecimiento & desarrollo , Células-Madre Neurales/citología , Animales , Animales Modificados Genéticamente , Encéfalo/citología , Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Linaje de la Célula , Drosophila/genética , Modelos Neurológicos , Células-Madre Neurales/clasificación , Células-Madre Neurales/metabolismo , NeurogénesisRESUMEN
Memory-retrieval processing of cross-modal sensory preconditioning is vital for understanding the plasticity underlying the interactions between modalities. As part of the sensory preconditioning paradigm, it has been hypothesized that the conditioned response to an unreinforced cue depends on the memory of the reinforced cue via a sensory link between the two cues. To test this hypothesis, we studied cross-modal memory-retrieval processing in a genetically tractable model organism, Drosophila melanogaster. By expressing the dominant temperature-sensitive shibire(ts1) (shi(ts1)) transgene, which blocks synaptic vesicle recycling of specific neural subsets with the Gal4/UAS system at the restrictive temperature, we specifically blocked visual and olfactory memory retrieval, either alone or in combination; memory acquisition remained intact for these modalities. Blocking the memory retrieval of the reinforced olfactory cues did not impair the conditioned response to the unreinforced visual cues or vice versa, in contrast to the canonical memory-retrieval processing of sensory preconditioning. In addition, these conditioned responses can be abolished by blocking the memory retrieval of the two modalities simultaneously. In sum, our results indicated that a conditioned response to an unreinforced cue in cross-modal sensory preconditioning can be recalled through parallel pathways.
Asunto(s)
Recuerdo Mental/fisiología , Vías Nerviosas/fisiología , Animales , Animales Modificados Genéticamente , Antígenos CD8/genética , Condicionamiento Psicológico/fisiología , Señales (Psicología) , Discriminación en Psicología/fisiología , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Femenino , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Memoria a Corto Plazo/fisiología , Modelos Biológicos , Cuerpos Pedunculados/citología , Cuerpos Pedunculados/fisiología , Neuronas/fisiología , Olfato/genética , Olfato/fisiología , Estadísticas no Paramétricas , Factores de Tiempo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Visión Ocular/genética , Visión Ocular/fisiologíaRESUMEN
Inflexible cognition and behavior are prominent features of prefrontal cortex damage and several neuropsychiatric disorders. The ability to flexibly adapt cognitive processing and behavior to dynamically changing environmental contingencies has been studied using the reversal learning paradigm in mammals, but the complexity of the brain circuits precludes a detailed analysis of the underlying neural mechanism. Here we study the neural circuitry mechanism supporting flexible behavior in a genetically tractable model organism, Drosophila melanogaster. Combining quantitative behavior analysis and genetic manipulation, we found that inhibition from a single pair of giant GABAergic neurons, the anterior paired lateral (APL) neurons, onto the mushroom bodies (MBs) selectively facilitates behavioral flexibility during visual reversal learning. This effect was mediated by ionotropic GABA(A) receptors in the MB. Moreover, flies with perturbed MB output recapitulated the poor reversal performance of flies with dysfunctional APL neurons. Importantly, we observed that flies with dysfunctional APL-MB circuit performed normally in simple forms of visual learning, including initial learning, extinction, and differential conditioning. Finally, we showed that acute disruption of the APL-MB circuit is sufficient to impair visual reversal learning. Together, these data suggest that the APL-MB circuit plays an essential role in the resolution of conflicting reinforcement contingencies and reveals an inhibitory neural mechanism underlying flexible behavior in Drosophila.
Asunto(s)
Neuronas GABAérgicas/fisiología , Sistema de la Línea Lateral/citología , Cuerpos Pedunculados/citología , Inhibición Neural/fisiología , Aprendizaje Inverso/fisiología , Percepción Visual , Análisis de Varianza , Animales , Animales Modificados Genéticamente , Conducta Animal/fisiología , Condicionamiento Psicológico , Drosophila , Proteínas de Drosophila/genética , Femenino , Vuelo Animal/fisiología , Regulación de la Expresión Génica/genética , Proteínas Fluorescentes Verdes/metabolismo , Sistema de la Línea Lateral/fisiología , Cuerpos Pedunculados/fisiología , Red Nerviosa/fisiología , Inhibición Neural/genética , Vías Nerviosas/fisiología , Orientación , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Tiempo de Reacción/genética , Tiempo de Reacción/fisiología , Transducción de Señal/fisiología , Estadísticas no Paramétricas , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Reversal learning has been widely used to probe the implementation of cognitive flexibility in the brain. Previous studies in monkeys identified an essential role of the orbitofrontal cortex (OFC) in reversal learning. However, the underlying circuits and molecular mechanisms are poorly understood. Here, we use the T-maze to investigate the neural mechanism of olfactory reversal learning in Drosophila. By adding a reversal training cycle to the classical learning protocol, we show that wild-type flies are able to reverse their choice according to the alteration of conditioned stimulus (CS)-unconditioned stimulus (US) contingency. The reversal protocol induced a specific suppression of the initial memory, an effect distinct from memory decay or extinction. GABA down-regulation in the anterior paired lateral (APL) neurons, which innervate the mushroom bodies (MBs), eliminates this suppression effect and impairs normal reversal. These findings reveal that inhibitory regulation from the GABAergic APL neurons facilitates olfactory reversal learning by suppressing initial memory in Drosophila.
Asunto(s)
Condicionamiento Clásico/fisiología , Neuronas GABAérgicas/fisiología , Sistema de la Línea Lateral/citología , Memoria/fisiología , Aprendizaje Inverso/fisiología , Olfato/fisiología , Animales , Animales Modificados Genéticamente , Regulación hacia Abajo/genética , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Dinaminas/genética , Dinaminas/metabolismo , Extinción Psicológica/fisiología , Glutamato Descarboxilasa/genética , Glutamato Descarboxilasa/metabolismo , Sistema de la Línea Lateral/fisiología , Cuerpos Pedunculados/citología , Cuerpos Pedunculados/fisiología , Mutación/genética , Inhibición Neural/genética , Inhibición Neural/fisiología , Odorantes , Interferencia de ARN/fisiología , Olfato/genética , Factores de Transcripción/genética , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Wiring a complex brain requires many neurons with intricate cell specificity, generated by a limited number of neural stem cells. Drosophila central brain lineages are a predetermined series of neurons, born in a specific order. To understand how lineage identity translates to neuron morphology, we mapped 18 Drosophila central brain lineages. While we found large aggregate differences between lineages, we also discovered shared patterns of morphological diversification. Lineage identity plus Notch-mediated sister fate govern primary neuron trajectories, whereas temporal fate diversifies terminal elaborations. Further, morphological neuron types may arise repeatedly, interspersed with other types. Despite the complexity, related lineages produce similar neuron types in comparable temporal patterns. Different stem cells even yield two identical series of dopaminergic neuron types, but with unrelated sister neurons. Together, these phenomena suggest that straightforward rules drive incredible neuronal complexity, and that large changes in morphology can result from relatively simple fating mechanisms.
Asunto(s)
Encéfalo/fisiología , Linaje de la Célula , Drosophila melanogaster/citología , Células-Madre Neurales/citología , Neurogénesis , Animales , Encéfalo/citología , Drosophila melanogaster/genética , Larva , Neuronas/citologíaRESUMEN
Building a sizable, complex brain requires both cellular expansion and diversification. One mechanism to achieve these goals is production of multiple transiently amplifying intermediate neural progenitors (INPs) from a single neural stem cell. Like mammalian neural stem cells, Drosophila type II neuroblasts utilize INPs to produce neurons and glia. Within a given lineage, the consecutively born INPs produce morphologically distinct progeny, presumably due to differential inheritance of temporal factors. To uncover the underlying temporal fating mechanisms, we profiled type II neuroblasts' transcriptome across time. Our results reveal opposing temporal gradients of Imp and Syp RNA-binding proteins (descending and ascending, respectively). Maintaining high Imp throughout serial INP production expands the number of neurons and glia with early temporal fate at the expense of cells with late fate. Conversely, precocious upregulation of Syp reduces the number of cells with early fate. Furthermore, we reveal that the transcription factor Seven-up initiates progression of the Imp/Syp gradients. Interestingly, neuroblasts that maintain initial Imp/Syp levels can still yield progeny with a small range of early fates. We therefore propose that the Seven-up-initiated Imp/Syp gradients create coarse temporal windows within type II neuroblasts to pattern INPs, which subsequently undergo fine-tuned subtemporal patterning.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Células-Madre Neurales/metabolismo , Proteínas de Unión al ARN/metabolismo , Receptores de Esteroides/metabolismo , Animales , Ciclo Celular , Linaje de la Célula , Proliferación Celular , Drosophila melanogaster/metabolismo , Perfilación de la Expresión Génica , Neurogénesis , Neuronas/citología , Neuronas/metabolismo , Factor de Células Madre/metabolismoRESUMEN
The morphology and physiology of neurons are directed by developmental decisions made within their lines of descent from single stem cells. Distinct stem cells may produce neurons having shared properties that define their cell class, such as the type of secreted neurotransmitter. The relationship between cell class and lineage is complex. Here we developed the transgenic cell class-lineage intersection (CLIn) system to assign cells of a particular class to specific lineages within the Drosophila brain. CLIn also enables birth-order analysis and genetic manipulation of particular cell classes arising from particular lineages. We demonstrated the power of CLIn in the context of the eight central brain type II lineages, which produce highly diverse progeny through intermediate neural progenitors. We mapped 18 dopaminergic neurons from three distinct clusters to six type II lineages that show lineage-characteristic neurite trajectories. In addition, morphologically distinct dopaminergic neurons are produced within a given lineage, and they arise in an invariant sequence. We also identified type II lineages that produce doublesex- and fruitless-expressing neurons and examined whether female-specific apoptosis in these lineages accounts for the lower number of these neurons in the female brain. Blocking apoptosis in these lineages resulted in more cells in both sexes with males still carrying more cells than females. This argues that sex-specific stem cell fate together with differential progeny apoptosis contribute to the final sexual dimorphism.
Asunto(s)
Encéfalo/fisiología , Linaje de la Célula , Drosophila melanogaster/fisiología , Animales , Animales Modificados Genéticamente , Apoptosis , Encéfalo/crecimiento & desarrollo , Drosophila melanogaster/crecimiento & desarrollo , Femenino , Masculino , Caracteres SexualesRESUMEN
Long-term tobacco use causes nicotine dependence via the regulation of a wide range of genes and is accompanied by various health problems. Studies in mammalian systems have revealed some key factors involved in the effects of nicotine, including nicotinic acetylcholine receptors (nAChRs), dopamine and other neurotransmitters. Nevertheless, the signaling pathways that link nicotine-induced molecular and behavioral modifications remain elusive. Utilizing a chronic nicotine administration paradigm, we found that adult male fruit flies exhibited locomotor hyperactivity after three consecutive days of nicotine exposure, while nicotine-naive flies did not. Strikingly, this chronic nicotine-induced locomotor hyperactivity (cNILH) was abolished in Decapping Protein 2 or 1 (Dcp2 or Dcp1) -deficient flies, while only Dcp2-deficient flies exhibited higher basal levels of locomotor activity than controls. These results indicate that Dcp2 plays a critical role in the response to chronic nicotine exposure. Moreover, the messenger RNA (mRNA) level of Dcp2 in the fly head was suppressed by chronic nicotine treatment, and up-regulation of Dcp2 expression in the nervous system blocked cNILH. These results indicate that down-regulation of Dcp2 mediates chronic nicotine-exposure-induced locomotor hyperactivity in Drosophila. The decapping proteins play a major role in mRNA degradation; however, their function in the nervous system has rarely been investigated. Our findings reveal a significant role for the mRNA decapping pathway in developing locomotor hyperactivity in response to chronic nicotine exposure and identify Dcp2 as a potential candidate for future research on nicotine dependence.