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

Deciphering where and how the information is acquired in the brain through learning and subsequently stored as memory constitutes one of the most challenging tasks in neuroscience¹. Neuroscientific research has led to the concept of a change in synaptic transmission as the neuronal substrate that underlies learning and memory formation^2,3. It is hypothesized that, during learning, synaptic connections between neuronal ensembles that are active during the perception of a stimulus become modified such that their combined activity pattern can be retrieved during memory recall, thereby instructing future behavioral action⁴. These "engram cells" and their synapses are often distributed across brain regions and levels of processing, which makes it difficult to assign observed changes in synaptic transmission to the learning of a task or a stimulus. To localize and visualize those synaptic changes that are causally linked to a specific learning task one needs an appropriate model system that allows for precisely confining those synapses.

For such an endeavor, Drosophila melanogaster is particularly suitable because it combines relative brain simplicity, behavioral richness, and experimental accessibility. Among the well-established model organisms, Drosophila is situated between the nematode C. elegans and genetically tractable mammals like mice in terms of neuronal complexity. The stereotypic number of neurons (~300) and limited behavioral repertoire is observed in C. elegans. Mammals, on the other hand, have millions of neurons and staggering behavioral complexity. The brain of the fruit fly is, with its ~100,000, neurons significantly smaller than the brains of most vertebrates, and many of the neurons are individually identifiable⁵. Yet, Drosophila demonstrate a broad spectrum of complex behaviors, including an ability to exhibit robust associative olfactory learning and memory formation, first described over 40 years ago⁶. In the course of this classical conditioning procedure, groups of flies are subjected to an odor as the conditioned stimulus (CS⁺) while they receive a punishing electric shock as the unconditioned stimulus (US). A second odor (CS^–) is then presented without any punishment. Thereby, the animals learn to avoid the odor associated with the punishment, which can be tested in a subsequent choice situation between the two odors, CS⁺ and CS^–. Work on dissecting the neuronal substrate underlying this behavior in Drosophila has identified the mushroom bodies (MB) as the primary site of the "engram"^7,8,9,10 and, therefore, the circuitry of this brain region was and is the subject of intense research in order to uncover the logic by which a memory engram is acquired and stored (recently reviewed in^11,12).

The Drosophila MB consists of ~2,000 intrinsic neurons (Kenyon cells) per hemisphere, organized in parallel axonal projections¹³. Axons of olfactory projection neurons are extended to the lateral protocerebra and to the MB calyces, the main dendritic input site of the MB and receive olfactory input from antennal lobes. The long, parallel axons bundle of Kenyon cells constitute the peduncle and the lobes. Most Kenyon cells bifurcate forming horizontal β/β'-lobes by extending one collateral towards the midline of the brain, and the vertical α/α'-lobes by extending second collateral projecting in the dorsal-anterior direction. The other group of Kenyon cells forms the horizontal γ-lobes¹³ of the MB where the learning process and subsequent short-term memory formation could be localized¹⁰. The MB lobes receive afferent input and provide efferent output, both of which are typically restricted to distinct compartmental sub-regions along the Kenyon cell axons^14,15,16. In particular, afferent dopaminergic MB input neurons have been shown to mediate value-based, e.g., punitive, reinforcing effects in associative olfactory learning^15,17. Stereotypic and individually identifiable efferent MB output neurons from the mushroom body lobes integrate information across large numbers of Kenyon cells, target diverse brain areas and bear behavior-instructive appetitive or aversive information¹⁵. This neuronal architecture has led to a concept of the organization of the associative engram. Odors are relatively precisely encoded by sparsely activated ensembles of Kenyon cells. The coincident activity of these Kenyon cell ensembles and release of dopamine – evoked by punishing stimuli – modulates transmission from Kenyon cell presynapses onto MB output neurons such that the animals will subsequently avoid this particular smell^10,12. We use this rather precisely defined and localized engram as a paradigmatic case to illustrate how these learning-dependent changes in synaptic activity can be determined and monitored.

The value of Drosophila as a model system relies strongly on the unmatched genetic toolbox that allows one to express transgenes for identifying, monitoring, and controlling single neurons within complex circuits¹⁸. The advent of techniques for neuronal activity monitoring – such as calcium imaging, discussed here – have allowed for the determination of neuronal activity patterns in response to a specific stimulus. By combining specific Gal4-driven expression of genetically encoded calcium indicators (GECIs) with olfactory stimulation, one can visualize the odor-evoked calcium dynamics of neurons of interest¹⁹. In this protocol, it is shown that by further coupling this technique with a classical conditioning paradigm, it is possible to examine these olfactory responses in the context of learning. Learning-induced plasticity can be further dissected using GECIs that are not only localized to a single specific neuron, but also to specific subcompartments of a neuron. Pech et al.²⁰ established a selection of tools that allow exactly this. By targeting GCaMP3²¹ to either the pre- or postsynapse – via linkage to the vertebrate Synaptophysin or dHomer, respectively²⁰– the differential modulation of these sites can be distinguished. This localization confers, in this context, an advantage over most GECIs that are ubiquitously present throughout the cytosol – e.g., GCaMP²², GCaMP3²¹, or GCaMP6²³ – because it means that pre- and postsynaptic transients can be distinguished from the overall integrated calcium influx that occurs as a result of neuron activation. This can provide clues about the location and types of plasticity that occur as a result of or that cause learning and memory formation. As an example, the protocol provided here shows the value of this tool in deciphering the modulation of MB output neurons during olfactory associative learning by targeting the expression of the calcium sensor to only the postsynapse. By monitoring, within an individual fly, odor-evoked activity before and after olfactory conditioning a direct comparison can be drawn between a naïve odor response and a learned odor response. Whilst fixed in the same imaging chamber, flies are exposed to a selection of odors. Then, they receive an aversive associative conditioning protocol in which one of these odors is paired with electric shock (becoming the CS⁺) and another odor is presented without reinforcement (becoming the CS^–). Finally, the flies are again exposed to the same odors as in the first step. Calcium dynamics are observed using two-photon microscopy.