André Fiala

Light-induced activation of distinct modulatory neurons triggers appetitive or aversive learning in Drosophila larvae

During classical conditioning, a positive or negative value is assigned to a previously neutral stimulus, thereby changing its significance for behavior. If an odor is associated with a negative stimulus, it can become repulsive. Conversely, an odor associated with a reward can become attractive. By using Drosophila larvae as a model system with minimal brain complexity, we address the question of which neurons attribute these values to odor stimuli. In insects, dopaminergic neurons are required for aversive learning, whereas octopaminergic neurons are necessary and sufficient for appetitive learning. However, it remains unclear whether two independent neuronal populations are sufficient to mediate such antagonistic values. We report the use of transgenically expressed channelrhodopsin-2, a light-activated cation channel, as a tool for optophysiological stimulation of genetically defined neuronal populations in Drosophila larvae. We demonstrate that distinct neuronal populations can be activated simply by illuminating the animals with blue light. Light-induced activation of dopaminergic neurons paired with an odor stimulus induces aversive memory formation, whereas activation of octopaminergic/tyraminergic neurons induces appetitive memory formation. These findings demonstrate that antagonistic modulatory subsystems are sufficient to substitute for aversive and appetitive reinforcement during classical conditioning.

Genetically Expressed Cameleon in Drosophila melanogaster Is Used to Visualize Olfactory Information in Projection Neurons

Complex external stimuli such as odorants are believed to be internally represented in the brain by spatiotemporal activity patterns of extensive neuronal ensembles. These activity patterns can be recorded by optical imaging techniques. However, optical imaging with conventional fluorescence dyes usually does not allow for resolving the activity of biologically defined groups of neurons. Therefore, specifically targeting reporter molecules to neuron populations of common genetic identity is an important goal. We report the use of the genetically encoded calcium-sensitive fluorescence protein cameleon 2.1 in the Drosophila brain. We visualized odorant-evoked intracellular calcium concentration changes in selectively labeled olfactory projection neurons both postsynaptically in the antennal lobe, the primary olfactory neuropil, and presynaptically in the mushroom body calyx, a structure involved in olfactory learning and memory. As a technical achievement, we show that calcium imaging with a genetically encoded fluorescence probe is feasible in a brain in vivo. This will allow one to combine Drosophilas advanced genetic tools with the physiological analysis of brain function. Moreover, we report for the first time optical imaging recordings in synaptic regions of the Drosophila mushroom body calyx and antennal lobe. This provides an important step for the use of Drosophila as a model system in olfaction.

Light Activation of an Innate Olfactory Avoidance Response in Drosophila

How specific sensory stimuli evoke specific behaviors is a fundamental problem in neurobiology. In Drosophila, most odorants elicit attraction or avoidance depending on their concentration, as well as their identity [1]. Such odorants, moreover, typically activate combinations of glomeruli in the antennal lobe of the brain [2-4], complicating the dissection of the circuits translating odor recognition into behavior. Carbon dioxide (CO2), in contrast, elicits avoidance over a wide range of concentrations [5, 6] and activates only a single glomerulus, V [5]. The V glomerulus receives projections from olfactory receptor neurons (ORNs) that coexpress two GPCRs, Gr21a and Gr63a, that together comprise a CO2 receptor [7-9]. These CO2-sensitive ORNs, located in the ab1 sensilla of the antenna, are called ab1c neurons [10]. Genetic silencing of ab1c neurons indicates that they are necessary for CO2-avoidance behavior [5]. Whether activation of these neurons alone is sufficient to elicit this behavior, or whether CO2 avoidance requires additional inputs (e.g., from the respiratory system), remains unclear. Here, we show that artificial stimulation of ab1c neurons with light (normally attractive to flies) elicits the avoidance behavior typical of CO2. Thus, avoidance behavior appears hardwired into the olfactory circuitry that detects CO2 in Drosophila.

Restoring polyamines protects from age-induced memory impairment in an autophagy-dependent manner

Age-dependent memory impairment is known to occur in several organisms, including Drosophila, mouse and human. However, the fundamental cellular mechanisms that underlie these impairments are still poorly understood, effectively hampering the development of pharmacological strategies to treat the condition. Polyamines are among the substances found to decrease with age in the human brain. We found that levels of polyamines (spermidine, putrescine) decreased in aging fruit flies, concomitant with declining memory abilities. Simple spermidine feeding not only restored juvenile polyamine levels, but also suppressed age-induced memory impairment. Ornithine decarboxylase-1, the rate-limiting enzyme for de novo polyamine synthesis, also protected olfactory memories in aged flies when expressed specifically in Kenyon cells, which are crucial for olfactory memory formation. Spermidine-fed flies showed enhanced autophagy (a form of cellular self-digestion), and genetic deficits in the autophagic machinery prevented spermidine-mediated rescue of memory impairments. Our findings indicate that autophagy is critical for suppression of memory impairments by spermidine and that polyamines, which are endogenously present, are candidates for pharmacological intervention.

Steroid-induced microRNA let-7 acts as a spatio-temporal code for neuronal cell fate in the developing Drosophila brain.

Mammalian neuronal stem cells produce multiple neuron types in the course of an individuals development. Similarly, neuronal progenitors in the Drosophila brain generate different types of closely related neurons that are born at specific time points during development. We found that in the post‐embryonic Drosophila brain, steroid hormones act as temporal cues that specify the cell fate of mushroom body (MB) neuroblast progeny. Chronological regulation of neurogenesis is subsequently mediated by the microRNA (miRNA) let‐7, absence of which causes learning impairment due to morphological MB defects. The miRNA let‐7 is required to regulate the timing of α′/β′ to α/β neuronal identity transition by targeting the transcription factor Abrupt. At a cellular level, the ecdysone‐let‐7‐Ab signalling pathway controls the expression levels of the cell adhesion molecule Fasciclin II in developing neurons that ultimately influences their differentiation. Our data propose a novel role for miRNAs as transducers between chronologically regulated developmental signalling and physical cell adhesion.

Channelrhodopsin-2–XXL, a powerful optogenetic tool for low-light applications

Significance Controlling neuronal activity in live tissue is a long sought-after goal in the neurosciences. Channelrhodopsin-2 (ChR2) is a microbial-type rhodopsin that can be genetically expressed to depolarize neurons with light. Thereby, this “optogenetic tool” delivers cellular specificity and elegant options for studying the neuronal basis of behavior in intact organisms. Unfortunately, low-light transmission through pigmented tissue greatly complicates light delivery to target cells and curtails experiments in freely moving animals. This study introduces a ChR mutant, ChR2-XXL, that gives rise to the largest photocurrents of all ChRs published so far and increases light sensitivity more than 10,000-fold over wild-type ChR2 in Drosophila larvae. As a result, behavioral photostimulation is evoked in freely moving flies using diffuse, ambient light. Channelrhodopsin-2 (ChR2) has provided a breakthrough for the optogenetic control of neuronal activity. In adult Drosophila melanogaster, however, its applications are severely constrained. This limitation in a powerful model system has curtailed unfolding the full potential of ChR2 for behavioral neuroscience. Here, we describe the D156C mutant, termed ChR2-XXL (extra high expression and long open state), which displays increased expression, improved subcellular localization, elevated retinal affinity, an extended open-state lifetime, and photocurrent amplitudes greatly exceeding those of all heretofore published ChR variants. As a result, neuronal activity could be efficiently evoked with ambient light and even without retinal supplementation. We validated the benefits of the variant in intact flies by eliciting simple and complex behaviors. We demonstrate efficient and prolonged photostimulation of monosynaptic transmission at the neuromuscular junction and reliable activation of a gustatory reflex pathway. Innate male courtship was triggered in male and female flies, and olfactory memories were written through light-induced associative training.

Presynapses in Kenyon Cell Dendrites in the Mushroom Body Calyx of Drosophila

Plastic changes at the presynaptic sites of the mushroom body (MB) principal neurons called Kenyon cells (KCs) are considered to represent a neuronal substrate underlying olfactory learning and memory. It is generally believed that presynaptic and postsynaptic sites of KCs are spatially segregated. In the MB calyx, KCs receive olfactory input from projection neurons (PNs) on their dendrites. Their presynaptic sites, however, are thought to be restricted to the axonal projections within the MB lobes. Here, we show that KCs also form presynapses along their calycal dendrites, by using novel transgenic tools for visualizing presynaptic active zones and postsynaptic densities. At these presynapses, vesicle release following stimulation could be observed. They reside at a distance from the PN input into the KC dendrites, suggesting that regions of presynaptic and postsynaptic differentiation are segregated along individual KC dendrites. KC presynapses are present in γ-type KCs that support short- and long-term memory in adult flies and larvae. They can also be observed in α/β-type KCs, which are involved in memory retrieval, but not in α′/β′-type KCs, which are implicated in memory acquisition and consolidation. We hypothesize that, as in mammals, recurrent activity loops might operate for memory retrieval in the fly olfactory system. The newly identified KC-derived presynapses in the calyx are, inter alia, candidate sites for the formation of memory traces during olfactory learning.

Optogenetically Induced Olfactory Stimulation in Drosophila Larvae Reveals the Neuronal Basis of Odor-Aversion behavior

Olfactory stimulation induces an odor-guided crawling behavior of Drosophila melanogaster larvae characterized by either an attractive or a repellent reaction. In order to understand the underlying processes leading to these orientations we stimulated single olfactory receptor neurons (ORNs) through photo-activation within an intact neuronal network. Using the Gal4-UAS system two light inducible proteins, the light-sensitive cation channel channelrhodopsin-2 (ChR-2) or the light-sensitive adenylyl cyclase (Pacα) were expressed in all or in individual ORNs of the larval olfactory system. Blue light stimulation caused an activation of these neurons, ultimately producing the illusion of an odor stimulus. Larvae were tested in a phototaxis assay for their orientation toward or away from the light source. Here we show that activation of Pacα expressing ORNs bearing the receptors Or33b or Or45a in blind norpA mutant larvae induces a repellent behavior away from the light. Conversely, photo-activation of the majority of ORNs induces attraction towards the light. Interestingly, in wild type larvae two ligands of Or33b and Or45a, octyl acetate and propionic ethylester, respectively, have been found to cause an escape reaction. Therefore, we combined light and odor stimulation to analyze the function of Or33b and Or45a expressing ORNs. We show that the larval olfactory system contains a designated neuronal pathway for repellent odorants and that activation of a specific class of ORNs already determines olfactory avoidance behavior.

The development of motor coordination in Drosophila embryos.

We used non-invasive muscle imaging to study the onset of motor activity and emergence of coordinated movement in Drosophila embryos. Earliest movements are myogenic, and neurally controlled muscle contractions first appear with the onset of bursting activity 17 hours after egg laying. Initial episodes of activity are poorly organised and coordinated crawling sequences only begin to appear after a further hour of bursting. Thus, network performance improves during this first period of activity. The embryo continues to exhibit bursts of crawling-like sequences until shortly before hatching, while other reflexes also mature. Bursting does not begin as a reflex response to sensory input but appears to reflect the onset of spontaneous activity in the motor network. It does not require GABA-mediated transmission, and, by using a light-activated channel to excite the network, we demonstrate activity-dependent depression that may cause burst termination.

Localization of the Contacts Between Kenyon Cells and Aminergic Neurons in the Drosophila melanogaster Brain Using SplitGFP Reconstitution

The mushroom body of the insect brain represents a neuronal circuit involved in the control of adaptive behavior, e.g., associative learning. Its function relies on the modulation of Kenyon cell activity or synaptic transmitter release by biogenic amines, e.g., octopamine, dopamine, or serotonin. Therefore, for a comprehensive understanding of the mushroom body, it is of interest not only to determine which modulatory neurons interact with Kenyon cells but also to pinpoint where exactly in the mushroom body they do so. To accomplish the latter, we made use of the GRASP technique and created transgenic Drosophila melanogaster that carry one part of a membrane‐bound splitGFP in Kenyon cells, along with a cytosolic red fluorescent marker. The second part of the splitGFP is expressed in distinct neuronal populations using cell‐specific Gal4 drivers. GFP is reconstituted only if these neurons interact with Kenyon cells in close proximity, which, in combination with two‐photon microscopy, provides a very high spatial resolution. We characterize spatially and microstructurally distinct contact regions between Kenyon cells and dopaminergic, serotonergic, and octopaminergic/tyraminergic neurons in all subdivisions of the mushroom body. Subpopulations of dopaminergic neurons contact complementary lobe regions densely. Octopaminergic/tyraminergic neurons contact Kenyon cells sparsely and are restricted mainly to the calyx, the α′‐lobes, and the γ‐lobes. Contacts of Kenyon cells with serotonergic neurons are heterogeneously distributed over the entire mushroom body. In summary, the technique enables us to localize precisely a segmentation of the mushroom body by differential contacts with aminergic neurons. J. Comp. Neurol. 521:3992–4026, 2013.