RESUMEN
Cryo-soft X-ray microscopy is an emerging imaging tool complementary to cryo-electron microscopy, allowing to image frozen hydrated specimens ten to hundred times thicker, but at lower resolution. We describe how the method, so far restricted to isolated small cells or cell monolayers, can be extended to large cells and tissue. We image the synapses of the Kenyon cells in frozen hydrated Drosophila brains combining cryo-soft X-ray microscopy of thick vitreous sections, and cryo-electron microscopy of ultrathin vitreous sections. We show how to obtain frozen hydrated sections of thicknesses ranging from 40 nm up to 2.5 µm, by tuning the sectioning speed of the cryo-microtome. A fluorescent stereo-microscope mounted on the cryo-microtome allowed us to target the regions of interest after GFP-labeling of synapses. Thick cryo-sections were imaged by cryo-soft X-ray microscopy at a resolution better than 25 nm, while ultrathin cryo-sections of the same regions were explored in parallel at the nanometre level of resolution by cryo-electron microscopy.
Asunto(s)
Encéfalo/ultraestructura , Microscopía por Crioelectrón/métodos , Drosophila/ultraestructura , Animales , Congelación , Secciones por Congelación/métodos , Rayos XRESUMEN
How does the concerted activity of neuronal populations shape behavior? Impediments to address this question are primarily due to critical experimental barriers. An integrated perspective on large scale neural information processing requires an in vivo approach that can combine the advantages of exhaustively observing all neurons dedicated to a given type of stimulus, and simultaneously achieve a resolution that is precise enough to capture individual neuron activity. Current experimental data from in vivo observations are either restricted to a small fraction of the total number of neurons, or are based on larger brain volumes but at a low spatial and temporal resolution. Consequently, fundamental questions as to how sensory information is represented on a population scale remain unanswered. In Drosophila melanogaster, the mushroom body (MB) represents an excellent model to analyze sensory coding and memory plasticity. In this work, we present an experimental setup coupled with a dedicated computational method that provides in vivo measurements of the activity of hundreds of densely packed somata uniformly spread in the MB. We exploit spinning-disk confocal 3D imaging over time of the whole MB cell body layer in vivo while it is exposed to olfactory stimulation. Importantly, to derive individual signal from densely packed somata, we have developed a fully automated image analysis procedure that takes advantage of the specificities of our data. After anisotropy correction, our approach operates a dedicated spot detection and registration over the entire time sequence to transform trajectories to identifiable clusters. This enabled us to discard spurious detections and reconstruct missing ones in a robust way. We demonstrate that this approach outperformed existing methods in this specific context and made possible high-throughput analysis of approximately 500 single somata uniformly spread over the MB in various conditions. Applying this approach, we find that learned experiences change the population code of odor representations in the MB. After long-term memory (LTM) formation, we quantified an increase in responsive somata count and a stable single neuron signal. We predict that this method, which should further enable studying the population pattern of neuronal activity, has the potential to uncover fine details of sensory processing and memory plasticity.
Asunto(s)
Calcio/metabolismo , Drosophila melanogaster/citología , Neuronas/metabolismo , Animales , Automatización , Memoria a Largo Plazo/fisiologíaRESUMEN
Non-caloric artificial sweeteners (NAS) are widely used in modern human food, raising the question about their health impact. Here we have asked whether NAS consumption is a neutral experience at neural and behavioral level, or if NAS can be interpreted and remembered as negative experience. We used behavioral and imaging approaches to demonstrate that Drosophila melanogaster learn the non-caloric property of NAS through post-ingestion process. These results show that sweet taste is predictive of an energy value, and its absence leads to the formation of what we call Caloric Frustration Memory (CFM) that devalues the NAS or its caloric enantiomer. CFM formation involves activity of the associative memory brain structure, the mushroom bodies (MBs). In vivo calcium imaging of MB-input dopaminergic neurons that respond to sugar showed a reduced response to NAS after CFM formation. Altogether, these findings demonstrate that NAS are a negative experience for the brain.
Asunto(s)
Conducta Animal/efectos de los fármacos , Drosophila melanogaster/fisiología , Memoria/efectos de los fármacos , Cuerpos Pedunculados/fisiología , Edulcorantes/efectos adversos , Animales , Calcio/química , Neuronas Dopaminérgicas/fisiología , Ingestión de Alimentos/psicología , Conducta Alimentaria/efectos de los fármacos , Femenino , Frustación , Masculino , Modelos Animales , Cuerpos Pedunculados/citología , Cuerpos Pedunculados/diagnóstico por imagen , Imagen Óptica/métodos , Gusto/fisiologíaRESUMEN
This paper reports on the spectral dynamics of perylene orange in thin sol-gel films. The studies are performed at the single molecule level to retrieve local information on such samples. The fluorescence spectrum of a molecule depends on the properties of the molecule itself and especially on its conformation in the ground state and in the state reached after excitation. Studies have been performed at room temperature and at a lower temperature, around 173 K. A large number of the recorded spectra reflect dual fluorescence. It is the rule at room temperature. However, at low temperature, single molecules either are relatively free to change conformation or are caught in a rigid environment. In the latter case, they present the spectrum of a rigid dye and we have identified the signature of several conformers of perylene orange in the ground state.
RESUMEN
We present an implementation of a sensorless adaptive optics loop in a widefield fluorescence microscope. This setup is designed to compensate for aberrations induced by the sample on both excitation and emission pathways. It allows fast optical sectioning inside a living Drosophila brain. We present a detailed characterization of the system performances. We prove that the gain brought to optical sectioning by realizing structured illumination microscopy with adaptive optics down to 50 µm deep inside living Drosophila brain.
Asunto(s)
Encéfalo/diagnóstico por imagen , Procesamiento de Imagen Asistido por Computador/métodos , Microscopía Fluorescente/métodos , Neuroimagen/métodos , Algoritmos , Animales , Animales Modificados Genéticamente , Química Encefálica , Drosophila , Diseño de Equipo , Proteínas Luminiscentes/química , Proteínas Luminiscentes/metabolismo , Microscopía Fluorescente/instrumentación , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
Sensory cues relevant to a food source, such as odors, can be associated with post-ingestion signals related either to food energetic value or toxicity. Despite numerous behavioral studies, a global understanding of the mechanisms underlying these long delay associations remains out of reach. Here, we demonstrate in Drosophila that the long-term association between an odor and a nutritious sugar depends on delayed post-ingestion signaling of energy level. We show at the neural circuit level that the activity of two pairs of dopaminergic neurons is necessary and sufficient to signal energy level to the olfactory memory center. Accordingly, we have identified in these dopaminergic neurons a delayed calcium trace that correlates with appetitive long-term memory formation. Altogether, these findings demonstrate that the Drosophila brain remembers food quality through a two-step mechanism that consists of the integration of olfactory and gustatory sensory information and then post-ingestion energetic value.
Asunto(s)
Dopamina/metabolismo , Drosophila/metabolismo , Memoria a Largo Plazo/fisiología , Animales , Encéfalo/fisiología , Neuronas Dopaminérgicas/metabolismo , Proteínas de Drosophila/antagonistas & inhibidores , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Metabolismo Energético , Glucosa/farmacología , Memoria a Largo Plazo/efectos de los fármacos , Interferencia de ARN , Transducción de Señal/efectos de los fármacos , Edulcorantes/farmacología , Factores de Transcripción/metabolismoRESUMEN
Aversive olfactory memory is formed in the mushroom bodies in Drosophila melanogaster. Memory retrieval requires mushroom body output, but the manner in which a memory trace in the mushroom body drives conditioned avoidance of a learned odor remains unknown. To identify neurons that are involved in olfactory memory retrieval, we performed an anatomical and functional screen of defined sets of mushroom body output neurons. We found that MB-V2 neurons were essential for retrieval of both short- and long-lasting memory, but not for memory formation or memory consolidation. MB-V2 neurons are cholinergic efferent neurons that project from the mushroom body vertical lobes to the middle superiormedial protocerebrum and the lateral horn. Notably, the odor response of MB-V2 neurons was modified after conditioning. As the lateral horn has been implicated in innate responses to repellent odorants, we propose that MB-V2 neurons recruit the olfactory pathway involved in innate odor avoidance during memory retrieval.
Asunto(s)
Recuerdo Mental/fisiología , Neuronas Motoras/fisiología , Cuerpos Pedunculados/citología , Olfato/fisiología , Vías Aferentes/fisiología , Análisis de Varianza , Animales , Animales Modificados Genéticamente , Reacción de Prevención/fisiología , Conducta Animal , Antígenos CD8/metabolismo , Colina O-Acetiltransferasa/metabolismo , Condicionamiento Psicológico , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Regulación de la Expresión Génica/fisiología , Glutamato Descarboxilasa/metabolismo , Proteínas Fluorescentes Verdes/genética , Memoria a Corto Plazo/fisiología , Modelos Biológicos , Neuronas Motoras/clasificación , Odorantes , Vías Olfatorias , Sinapsinas/metabolismo , Temperatura , Factores de Tiempo , Factores de Transcripción/metabolismo , Tirosina 3-Monooxigenasa/metabolismo , Proteínas de Transporte Vesicular de Glutamato/metabolismoRESUMEN
The dynamics of PKA activity in the olfactory learning and memory center, the mushroom bodies (MBs), are still poorly understood. We addressed this issue in vivo using a PKA FRET probe. Application of dopamine, the main neuromodulator involved in aversive learning, resulted in PKA activation specifically in the vertical lobe, whereas octopamine, involved in appetitive learning, stimulated PKA in all MB lobes. Strikingly, MB lobes were homogeneously activated by dopamine in the learning mutant dunce, showing that Dunce phosphodiesterase plays a major role in the spatial regulation of cAMP dynamics. Furthermore, costimulation with acetylcholine and either dopamine or octopamine led to a synergistic activation of PKA in the MBs that depends on Rutabaga adenylyl cyclase. Our results suggest that Rutabaga acts as a coincidence detector and demonstrate the existence of subcellular domains of PKA activity that could underlie the functional specialization of MB lobes in aversive and appetitive learning.
Asunto(s)
Adenilil Ciclasas/metabolismo , Aprendizaje por Asociación/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas de Drosophila/metabolismo , Memoria a Corto Plazo/fisiología , Cuerpos Pedunculados/metabolismo , Acetilcolina/metabolismo , Acetilcolina/farmacología , Adenilil Ciclasas/genética , Animales , Animales Modificados Genéticamente , Aprendizaje por Asociación/efectos de los fármacos , Reacción de Prevención/efectos de los fármacos , Reacción de Prevención/fisiología , Axones/efectos de los fármacos , Axones/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Dendritas/efectos de los fármacos , Dendritas/metabolismo , Dopamina/metabolismo , Dopamina/farmacología , Proteínas de Drosophila/genética , Drosophila melanogaster , Procesamiento de Imagen Asistido por Computador , Memoria a Corto Plazo/efectos de los fármacos , Microscopía de Fluorescencia por Excitación Multifotónica , Motivación/efectos de los fármacos , Motivación/fisiología , Cuerpos Pedunculados/efectos de los fármacos , Octopamina/metabolismo , Octopamina/fisiología , Vías Olfatorias/efectos de los fármacos , Vías Olfatorias/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Mutations in the human neurotrypsin gene are associated with autosomal recessive mental retardation. To further understand the pathophysiological consequences of the lack of this serine protease, we studied Tequila (Teq), the Drosophila neurotrypsin ortholog, using associative memory as a behavioral readout. We found that teq inactivation resulted in a long-term memory (LTM)-specific defect. After LTM conditioning of wild-type flies, teq expression transiently increased in the mushroom bodies. Moreover, specific inhibition of teq expression in adult mushroom bodies resulted in a reversible LTM defect. Hence, the Teq pathway is essential for information processing in Drosophila.