RESUMEN
The ability to optically image cellular transmembrane voltages at millisecond-timescale resolutions can offer unprecedented insight into the function of living brains in behaving animals. Here, we present a point mutation that increases the sensitivity of Ace2 opsin-based voltage indicators. We use the mutation to develop Voltron2, an improved chemigeneic voltage indicator that has a 65% higher sensitivity to single APs and 3-fold higher sensitivity to subthreshold potentials than Voltron. Voltron2 retained the sub-millisecond kinetics and photostability of its predecessor, although with lower baseline fluorescence. In multiple in vitro and in vivo comparisons with its predecessor across multiple species, we found Voltron2 to be more sensitive to APs and subthreshold fluctuations. Finally, we used Voltron2 to study and evaluate the possible mechanisms of interneuron synchronization in the mouse hippocampus. Overall, we have discovered a generalizable mutation that significantly increases the sensitivity of Ace2 rhodopsin-based sensors, improving their voltage reporting capability.
Asunto(s)
Enzima Convertidora de Angiotensina 2 , Rodopsina , Ratones , Animales , Potenciales de Acción/fisiología , Rodopsina/genética , Neuronas/fisiología , Mutación/genéticaRESUMEN
Visual systems can exploit spatial correlations in the visual scene by using retinotopy, the organizing principle by which neighboring cells encode neighboring spatial locations. However, retinotopy is often lost, such as when visual pathways are integrated with other sensory modalities. How is spatial information processed outside of strictly visual brain areas? Here, we focused on visual looming responsive LC6 cells in Drosophila, a population whose dendrites collectively cover the visual field, but whose axons form a single glomerulus-a structure without obvious retinotopic organization-in the central brain. We identified multiple cell types downstream of LC6 in the glomerulus and found that they more strongly respond to looming in different portions of the visual field, unexpectedly preserving spatial information. Through EM reconstruction of all LC6 synaptic inputs to the glomerulus, we found that LC6 and downstream cell types form circuits within the glomerulus that enable spatial readout of visual features and contralateral suppression-mechanisms that transform visual information for behavioral control.
Asunto(s)
Encéfalo/fisiología , Neuronas/fisiología , Vías Visuales/fisiología , Percepción Visual/fisiología , Animales , Drosophila melanogasterRESUMEN
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.
Asunto(s)
Calcio/análisis , Colorantes Fluorescentes/química , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Animales , Drosophila , Femenino , Rayos Láser , Masculino , Ratones , Ratones Endogámicos C57BL , Imagen Molecular , Corteza Somatosensorial/químicaRESUMEN
Identifying the neurotransmitters used by specific neurons is a critical step in understanding the function of neural circuits. However, methods for the consistent and efficient detection of neurotransmitter markers remain limited. Fluorescence in situ hybridization (FISH) enables direct labeling of type-specific mRNA in neurons. Recent advances in FISH allow this technique to be carried out in intact tissue samples such as whole-mount Drosophila melanogaster brains. Here, we present a FISH platform for high-throughput detection of eight common neurotransmitter phenotypes in Drosophila brains. We greatly increase FISH throughput by processing samples mounted on coverslips and optimizing fluorophore choice for each probe to facilitate multiplexing. As application examples, we demonstrate cases of neurotransmitter coexpression, reveal neurotransmitter phenotypes of specific cell types, and explore the onset of neurotransmitter expression in the developing optic lobe. Beyond neurotransmitter markers, our protocols can in principle be used for large-scale FISH detection of any mRNA in whole-mount fly brains.
Asunto(s)
Encéfalo/metabolismo , Hibridación Fluorescente in Situ/métodos , Neurotransmisores/metabolismo , Animales , Química Encefálica , Drosophila/metabolismo , Neurotransmisores/análisisRESUMEN
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.
Asunto(s)
Calcio/metabolismo , Optogenética/métodos , Animales , Señalización del Calcio/fisiología , Drosophila , Cinética , Neuronas/metabolismo , Imagen Óptica/métodosRESUMEN
In most animals, the brain controls the body via a set of descending neurons (DNs) that traverse the neck. DN activity activates, maintains or modulates locomotion and other behaviors. Individual DNs have been well-studied in species from insects to primates, but little is known about overall connectivity patterns across the DN population. We systematically investigated DN anatomy in Drosophila melanogaster and created over 100 transgenic lines targeting individual cell types. We identified roughly half of all Drosophila DNs and comprehensively map connectivity between sensory and motor neuropils in the brain and nerve cord, respectively. We find the nerve cord is a layered system of neuropils reflecting the fly's capability for two largely independent means of locomotion -- walking and flight -- using distinct sets of appendages. Our results reveal the basic functional map of descending pathways in flies and provide tools for systematic interrogation of neural circuits.
Asunto(s)
Encéfalo/fisiología , Drosophila melanogaster/fisiología , Vías Eferentes/fisiología , Locomoción/fisiología , Neuronas/fisiología , Neurópilo/fisiología , Animales , Animales Modificados Genéticamente , Encéfalo/anatomía & histología , Encéfalo/citología , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/anatomía & histología , Drosophila melanogaster/citología , Genes Reporteros , Neuronas/citología , Neurópilo/citología , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The behavioral state of an animal can dynamically modulate visual processing. In flies, the behavioral state is known to alter the temporal tuning of neurons that carry visual motion information into the central brain. However, where this modulation occurs and how it tunes the properties of this neural circuit are not well understood. Here, we show that the behavioral state alters the baseline activity levels and the temporal tuning of the first directionally selective neuron in the ON motion pathway (T4) as well as its primary input neurons (Mi1, Tm3, Mi4, Mi9). These effects are especially prominent in the inhibitory neuron Mi4, and we show that central octopaminergic neurons provide input to Mi4 and increase its excitability. We further show that octopamine neurons are required for sustained behavioral responses to fast-moving, but not slow-moving, visual stimuli in walking flies. These results indicate that behavioral-state modulation acts directly on the inputs to the directionally selective neurons and supports efficient neural coding of motion stimuli.
Asunto(s)
Conducta Animal/fisiología , Actividad Motora/fisiología , Neuronas/metabolismo , Octopamina/metabolismo , Animales , Drosophila , Neuronas/citologíaRESUMEN
Diffuse neuromodulatory systems such as norepinephrine (NE) control brain-wide states such as arousal, but whether they control complex social behaviors more specifically is not clear. Octopamine (OA), the insect homolog of NE, is known to promote both arousal and aggression. We have performed a systematic, unbiased screen to identify OA receptor-expressing neurons (OARNs) that control aggression in Drosophila. Our results uncover a tiny population of male-specific aSP2 neurons that mediate a specific influence of OA on aggression, independent of any effect on arousal. Unexpectedly, these neurons receive convergent input from OA neurons and P1 neurons, a population of FruM+ neurons that promotes male courtship behavior. Behavioral epistasis experiments suggest that aSP2 neurons may constitute an integration node at which OAergic neuromodulation can bias the output of P1 neurons to favor aggression over inter-male courtship. These results have potential implications for thinking about the role of related neuromodulatory systems in mammals.
Asunto(s)
Agresión/fisiología , Proteínas de Drosophila/fisiología , Drosophila/citología , Drosophila/fisiología , Vías Nerviosas , Neuronas/fisiología , Receptores de Neurotransmisores/fisiología , Conducta Social , Animales , Animales Modificados Genéticamente , Nivel de Alerta/fisiología , Cortejo , Proteínas de Drosophila/genética , Interneuronas/fisiología , Masculino , Receptores de Neurotransmisores/genéticaRESUMEN
We describe a fluorescence in situ hybridization method that permits detection of the localization and abundance of single mRNAs (smFISH) in cleared whole-mount adult Drosophila brains. The approach is rapid and multiplexable and does not require molecular amplification; it allows facile quantification of mRNA expression with subcellular resolution on a standard confocal microscope. We further demonstrate single-mRNA detection across the entire brain using a custom Bessel beam structured illumination microscope (BB-SIM).
Asunto(s)
Encéfalo/metabolismo , Drosophila/metabolismo , Hibridación Fluorescente in Situ/métodos , Microscopía/métodos , ARN Mensajero/metabolismo , Animales , Drosophila/anatomía & histología , Microscopía/instrumentaciónRESUMEN
The perception of visual motion is critical for animal navigation, and flies are a prominent model system for exploring this neural computation. In Drosophila, the T4 cells of the medulla are directionally selective and necessary for ON motion behavioral responses. To examine the emergence of directional selectivity, we developed genetic driver lines for the neuron types with the most synapses onto T4 cells. Using calcium imaging, we found that these neuron types are not directionally selective and that selectivity arises in the T4 dendrites. By silencing each input neuron type, we identified which neurons are necessary for T4 directional selectivity and ON motion behavioral responses. We then determined the sign of the connections between these neurons and T4 cells using neuronal photoactivation. Our results indicate a computational architecture for motion detection that is a hybrid of classic theoretical models.
Asunto(s)
Potenciales de Acción/fisiología , Dendritas/fisiología , Bulbo Raquídeo/fisiología , Percepción de Movimiento/fisiología , Neuronas/fisiología , Vías Visuales/fisiología , Animales , Calcio/metabolismo , Drosophila , Perfilación de la Expresión Génica , Bulbo Raquídeo/citología , Modelos NeurológicosRESUMEN
It is unclear how regulatory genes establish neural circuits that compose sex-specific behaviors. The Drosophila melanogaster male courtship song provides a powerful model to study this problem. Courting males vibrate a wing to sing bouts of pulses and hums, called pulse and sine song, respectively. We report the discovery of male-specific thoracic interneurons-the TN1A neurons-that are required specifically for sine song. The TN1A neurons can drive the activity of a sex-non-specific wing motoneuron, hg1, which is also required for sine song. The male-specific connection between the TN1A neurons and the hg1 motoneuron is regulated by the sexual differentiation gene doublesex. We find that doublesex is required in the TN1A neurons during development to increase the density of the TN1A arbors that interact with dendrites of the hg1 motoneuron. Our findings demonstrate how a sexual differentiation gene can build a sex-specific circuit motif by modulating neuronal arborization.
Asunto(s)
Cortejo , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiología , Red Nerviosa/metabolismo , Vocalización Animal/fisiología , Animales , Dendritas/metabolismo , Femenino , Masculino , Neuronas Motoras/metabolismoRESUMEN
Optogenetics allows the manipulation of neural activity in freely moving animals with millisecond precision, but its application in Drosophila melanogaster has been limited. Here we show that a recently described red activatable channelrhodopsin (ReaChR) permits control of complex behavior in freely moving adult flies, at wavelengths that are not thought to interfere with normal visual function. This tool affords the opportunity to control neural activity over a broad dynamic range of stimulation intensities. Using time-resolved activation, we show that the neural control of male courtship song can be separated into (i) probabilistic, persistent and (ii) deterministic, command-like components. The former, but not the latter, neurons are subject to functional modulation by social experience, which supports the idea that they constitute a locus of state-dependent influence. This separation is not evident using thermogenetic tools, a result underscoring the importance of temporally precise control of neuronal activation in the functional dissection of neural circuits in Drosophila.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila/fisiología , Optogenética , Rodopsina/metabolismo , Conducta Sexual Animal/fisiología , Animales , Drosophila/genética , Proteínas de Drosophila/genética , Femenino , Masculino , Neuronas/fisiología , Estimulación Luminosa , Percepción del GustoRESUMEN
Insects show sophisticated odor-mediated behaviors controlled by an olfactory system that is genetically and anatomically simpler than that of vertebrates, providing an attractive system to investigate the mechanistic link between behavior and odor perception. Advances in neuroscience have been facilitated by modern optical imaging technologies--both in instrumentation and in probe design--that permit the visualization of functional neural circuits. Imaging calcium activity in genetically defined populations of neurons provides an important tool for investigating the function of neural circuits. This article describes a two-photon imaging system for monitoring neural activity in the Drosophila antennal lobe. Odor-evoked calcium activity is followed by measuring the specific expression of the calcium-sensitive green fluorescent protein G-CaMP in Drosophila antennae-brain preparations.
Asunto(s)
Calcio/análisis , Drosophila/fisiología , Imagen Óptica/métodos , Animales , Conducta Animal , Vías Olfatorias/fisiología , Percepción Olfatoria , Imagen Óptica/instrumentación , OlfatoRESUMEN
Stroking of the skin produces pleasant sensations that can occur during social interactions with conspecifics, such as grooming. Despite numerous physiological studies (reviewed in ref. 2), molecularly defined sensory neurons that detect pleasant stroking of hairy skin in vivo have not been reported. Previously, we identified a rare population of unmyelinated sensory neurons in mice that express the G-protein-coupled receptor MRGPRB4 (refs 5, 6). These neurons exclusively innervate hairy skin with large terminal arborizations that resemble the receptive fields of C-tactile (CT) afferents in humans. Unlike other molecularly defined mechanosensory C-fibre subtypes, MRGPRB4(+) neurons could not be detectably activated by sensory stimulation of the skin ex vivo. Therefore, we developed a preparation for calcium imaging in the spinal projections of these neurons during stimulation of the periphery in intact mice. Here we show that MRGPRB4(+) neurons are activated by massage-like stroking of hairy skin, but not by noxious punctate mechanical stimulation. By contrast, a different population of C fibres expressing MRGPRD was activated by pinching but not by stroking, consistent with previous physiological and behavioural data. Pharmacogenetic activation of Mrgprb4-expressing neurons in freely behaving mice promoted conditioned place preference, indicating that such activation is positively reinforcing and/or anxiolytic. These data open the way to understanding the function of MRGPRB4 neurons during natural behaviours, and provide a general approach to the functional characterization of genetically identified subsets of somatosensory neurons in vivo.
Asunto(s)
Fibras Nerviosas Amielínicas/metabolismo , Receptores Acoplados a Proteínas G/genética , Piel/inervación , Tacto/genética , Animales , Perfilación de la Expresión Génica , Ratones , Receptores Acoplados a Proteínas G/metabolismo , Células Receptoras Sensoriales/metabolismoRESUMEN
Behavior cannot be predicted from a "connectome" because the brain contains a chemical "map" of neuromodulation superimposed upon its synaptic connectivity map. Neuromodulation changes how neural circuits process information in different states, such as hunger or arousal. Here we describe a genetically based method to map, in an unbiased and brain-wide manner, sites of neuromodulation under different conditions in the Drosophila brain. This method, and genetic perturbations, reveal that the well-known effect of hunger to enhance behavioral sensitivity to sugar is mediated, at least in part, by the release of dopamine onto primary gustatory sensory neurons, which enhances sugar-evoked calcium influx. These data reinforce the concept that sensory neurons constitute an important locus for state-dependent gain control of behavior and introduce a methodology that can be extended to other neuromodulators and model organisms.
Asunto(s)
Dopamina/metabolismo , Drosophila melanogaster/fisiología , Neurotransmisores/metabolismo , Transducción de Señal , Animales , Regulación del Apetito , Arrestina/metabolismo , Encéfalo/fisiología , Mapeo Encefálico/métodos , Conducta Alimentaria , Femenino , Receptores Dopaminérgicos/metabolismo , Células Receptoras Sensoriales/metabolismoRESUMEN
Investigating how information propagates between layers in the olfactory system is an important step toward understanding the olfactory code. Each glomerular output projection neuron (PN) receives two sources of input: the olfactory receptor neurons (ORNs) of the same glomerulus and interneurons that innervate many glomeruli. We therefore asked how these inputs interact to produce PN output. We used receptor gene mutations to silence all of the ORNs innervating a specific glomerulus and recorded PN activity with two-photon calcium imaging and electrophysiology. We found evidence for balanced excitatory and inhibitory synaptic inputs but saw little or no response in the absence of direct ORN input. We next asked whether any transformation of activity occurs at successive layers of the antennal lobe. We found a strong link between PN firing and dendritic calcium elevation, the latter of which is tightly correlated with calcium activity in ORN axons, supporting the idea of glomerular propagation of olfactory information. Finally, we showed that odors are represented by a sparse population of PNs. Together, these results are consistent with the idea that direct receptor input provides the main excitatory drive to PNs, whereas interneurons modulate PN output. Balanced excitatory and inhibitory interneuron input may provide a mechanism to adjust PN sensitivity.
Asunto(s)
Drosophila/citología , Drosophila/fisiología , Neuronas Receptoras Olfatorias/fisiología , Olfato/fisiología , Potenciales de Acción/fisiología , Animales , Encéfalo/citología , Encéfalo/metabolismo , Encéfalo/fisiología , Calcio/metabolismo , Drosophila/metabolismo , Interneuronas/metabolismo , Interneuronas/fisiología , Neuronas Receptoras Olfatorias/metabolismo , Técnicas de Placa-Clamp , Olfato/genéticaRESUMEN
All animals exhibit innate behaviours in response to specific sensory stimuli that are likely to result from the activation of developmentally programmed neural circuits. Here we observe that Drosophila exhibit robust avoidance to odours released by stressed flies. Gas chromatography and mass spectrometry identifies one component of this 'Drosophila stress odorant (dSO)' as CO2. CO2 elicits avoidance behaviour, at levels as low as 0.1%. We used two-photon imaging with the Ca2+-sensitive fluorescent protein G-CaMP to map the primary sensory neurons governing avoidance to CO2. CO2 activates only a single glomerulus in the antennal lobe, the V glomerulus; moreover, this glomerulus is not activated by any of 26 other odorants tested. Inhibition of synaptic transmission in sensory neurons that innervate the V glomerulus, using a temperature-sensitive Shibire gene (Shi(ts)), blocks the avoidance response to CO2. Inhibition of synaptic release in the vast majority of other olfactory receptor neurons has no effect on this behaviour. These data demonstrate that the activation of a single population of sensory neurons innervating one glomerulus is responsible for an innate avoidance behaviour in Drosophila.
Asunto(s)
Reacción de Prevención/fisiología , Drosophila melanogaster/fisiología , Instinto , Neuronas Receptoras Olfatorias/citología , Neuronas Receptoras Olfatorias/fisiología , Aire/análisis , Animales , Reacción de Prevención/efectos de los fármacos , Encéfalo/citología , Encéfalo/efectos de los fármacos , Encéfalo/fisiología , Calcio/metabolismo , Dióxido de Carbono/análisis , Dióxido de Carbono/farmacología , Drosophila melanogaster/citología , Drosophila melanogaster/efectos de los fármacos , Hidroxiurea/farmacología , Ratones , Odorantes/análisis , Neuronas Receptoras Olfatorias/efectos de los fármacos , Estrés Fisiológico/fisiopatologíaRESUMEN
An understanding of the logic of odor perception requires a functional analysis of odor-evoked patterns of activity in neural assemblies in the brain. We have developed a sensitive imaging system in the Drosophila brain that couples two-photon microscopy with the specific expression of the calcium-sensitive fluorescent protein, G-CaMP. At natural odor concentration, each odor elicits a distinct and sparse spatial pattern of activity in the antennal lobe that is conserved in different flies. Patterns of glomerular activity are similar upon imaging of sensory and projection neurons, suggesting the faithful transmission of sensory input to higher brain centers. Finally, we demonstrate that the response pattern of a given glomerulus is a function of the specificity of a single odorant receptor. The development of this imaging system affords an opportunity to monitor activity in defined neurons throughout the fly brain with high sensitivity and excellent spatial resolution.
Asunto(s)
Encéfalo/fisiología , Calcio/metabolismo , Drosophila/fisiología , Odorantes , Vías Olfatorias/fisiología , Olfato/fisiología , Animales , Axones/metabolismo , Encéfalo/anatomía & histología , Encéfalo/citología , Señalización del Calcio , Dendritas/metabolismo , Diagnóstico por Imagen/métodos , Drosophila/anatomía & histología , Drosophila/citología , Ganglios de Invertebrados/citología , Ganglios de Invertebrados/metabolismo , Vías Olfatorias/citología , Neuronas Receptoras Olfatorias/citología , Neuronas Receptoras Olfatorias/metabolismo , FotonesRESUMEN
In the fruit fly, Drosophila, olfactory sensory neurons expressing a given receptor project to spatially invariant loci in the antennal lobe to create a topographic map of receptor activation. We have asked how the map in the antennal lobe is represented in higher sensory centers in the brain. Random labeling of individual projection neurons using the FLP-out technique reveals that projection neurons that innervate the same glomerulus exhibit strikingly similar axonal topography, whereas neurons from different glomeruli display very different patterns of projection in the protocerebrum. These results demonstrate that a topographic map of olfactory information is retained in higher brain centers, but the character of the map differs from that of the antennal lobe, affording an opportunity for integration of olfactory sensory input.