Opposing GPCR signaling programs protein intake setpoint in Drosophila.

Animals defend a target level for their fundamental needs, including food, water, and sleep. Deviation from the target range, or "setpoint," triggers motivated behaviors to eliminate that difference. Whether and how the setpoint itself is encoded remains enigmatic for all motivated behaviors. Employing a high-throughput feeding assay in Drosophila, we demonstrate that the protein intake setpoint is set to different values in male, virgin female, and mated female flies to meet their varying protein demands. Leveraging this setpoint variability, we found, remarkably, that the information on the intake setpoint is stored within the protein hunger neurons as the resting membrane potential. Two RFamide G protein-coupled receptor (GPCR) pathways, by tuning the resting membrane potential in opposite directions, coordinately program and adjust the protein intake setpoint. Together, our studies map the protein intake setpoint to a single trackable physiological parameter and elucidate the cellular and molecular mechanisms underlying setpoint determination and modulation.

Visualization of learning-induced synaptic plasticity in output neurons of the Drosophila mushroom body γ-lobe.

By learning, through experience, which stimuli coincide with dangers, it is possible to predict outcomes and act pre-emptively to ensure survival. In insects, this process is localized to the mushroom body (MB), the circuitry of which facilitates the coincident detection of sensory stimuli and punishing or rewarding cues and, downstream, the execution of appropriate learned behaviors. Here, we focused our attention on the mushroom body output neurons (MBONs) of the γ-lobes that act as downstream synaptic partners of the MB γ-Kenyon cells (KCs) to ask how the output of the MB γ-lobe is shaped by olfactory associative conditioning, distinguishing this from non-associative stimulus exposure effects, and without the influence of downstream modulation. This was achieved by employing a subcellularly localized calcium sensor to specifically monitor activity at MBON postsynaptic sites. Therein, we identified a robust associative modulation within only one MBON postsynaptic compartment (MBON-γ1pedc > α/ß), which displayed a suppressed postsynaptic response to an aversively paired odor. While this MBON did not undergo non-associative modulation, the reverse was true across the remainder of the γ-lobe, where general odor-evoked adaptation was observed, but no conditioned odor-specific modulation. In conclusion, associative synaptic plasticity underlying aversive olfactory learning is localized to one distinct synaptic γKC-to-γMBON connection.

Visualization of naive and learned odor representations using in vivo calcium imaging and immunohistochemical bouton mapping of single Drosophila mushroom body neurons.

This protocol enables the quantification of odor-evoked calcium activity in mushroom body Kenyon cells of the Drosophila melanogaster brain at the single bouton level. We also present subsequent characterization of naive and learned odor representations in the context of olfactory coding. This approach to analyzing the neuronal basis of associative learning provides a substrate for similar studies, perhaps in other animals, to probe the attributes of a neuronal memory trace at the level of synapses distributed across neurons. For complete details on the use and execution of this protocol, please refer to Bilz et al. (2020).

Visualization of a Distributed Synaptic Memory Code in the Drosophila Brain.

During associative conditioning, animals learn which sensory cues are predictive for positive or negative conditions. Because sensory cues are encoded by distributed neurons, one has to monitor plasticity across many synapses to capture how learned information is encoded. We analyzed synaptic boutons of Kenyon cells of the Drosophila mushroom body γ lobe, a brain structure that mediates olfactory learning. A fluorescent Ca2+ sensor was expressed in single Kenyon cells so that axonal boutons could be assigned to distinct cells and Ca2+ could be measured across many animals. Learning induced directed synaptic plasticity in specific compartments along the axons. Moreover, we show that odor-evoked Ca2+ dynamics across boutons decorrelate as a result of associative learning. Information theory indicates that learning renders the stimulus representation more distinct compared with naive stimuli. These data reveal that synaptic boutons rather than cells act as individually modifiable units, and coherence among them is a memory-encoding parameter.

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster.

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. Learning-induced changes in synaptic transmission are often distributed across many neurons and levels of processing in the brain. Therefore, methods to visualize learning-dependent synaptic plasticity across neurons are needed. The fruit fly Drosophila melanogaster represents a particularly favorable model organism to study neuronal circuits underlying learning. The protocol presented here demonstrates a way in which the processes underlying the formation of associative olfactory memories, i.e., synaptic activity and their changes, can be monitored in vivo. Using the broad array of genetic tools available in Drosophila, it is possible to specifically express genetically encoded calcium indicators in determined cell populations and even single cells. By fixing a fly in place, and opening the head capsule, it is possible to visualize calcium dynamics in these cells whilst delivering olfactory stimuli. Additionally, we demonstrate a set-up in which the fly can be subjected, simultaneously, to electric shocks to the body. This provides a system in which flies can undergo classical olfactory conditioning - whereby a previously naïve odor is learned to be associated with electric shock punishment - at the same time as the representation of this odor (and other untrained odors) is observed in the brain via two-photon microscopy. Our lab has previously reported the generation of synaptically localized calcium sensors, which enables one to confine the fluorescent calcium signals to pre- or postsynaptic compartments. Two-photon microscopy provides a way to spatially resolve fine structures. We exemplify this by focusing on neurons integrating information from the mushroom body, a higher-order center of the insect brain. Overall, this protocol provides a method to examine the synaptic connections between neurons whose activity is modulated as a result of olfactory learning.