Mapping Kenyon cell inputs in Drosophila using dye electroporation.

Here, we describe a technique for charting the inputs of individual Kenyon cells in the Drosophila brain. In this technique, a single Kenyon cell per brain hemisphere is photo-labeled to visualize its claw-like dendritic terminals; a dye-filled electrode is used to backfill the projection neuron connected to each claw. This process can be repeated in hundreds of brains to build a connectivity matrix. Statistical analyses of such a matrix can reveal connectivity patterns such as random input and biased connectivity. For complete details on the use and execution of this protocol, please refer to Hayashi et al. (2022).1.

Evolution of connectivity architecture in the Drosophila mushroom body.

Brain evolution has primarily been studied at the macroscopic level by comparing the relative size of homologous brain centers between species. How neuronal circuits change at the cellular level over evolutionary time remains largely unanswered. Here, using a phylogenetically informed framework, we compare the olfactory circuits of three closely related Drosophila species that differ radically in their chemical ecology: the generalists Drosophila melanogaster and Drosophila simulans that feed on fermenting fruit, and Drosophila sechellia that specializes on ripe noni fruit. We examine a central part of the olfactory circuit that has not yet been investigated in these species - the connections between the projection neurons of the antennal lobe and the Kenyon cells of the mushroom body, an associative brain center - to identify species-specific connectivity patterns. We found that neurons encoding food odors - the DC3 neurons in D. melanogaster and D. simulans and the DL2d neurons in D. sechellia - connect more frequently with Kenyon cells, giving rise to species-specific biases in connectivity. These species-specific differences in connectivity reflect two distinct neuronal phenotypes: in the number of projection neurons or in the number of presynaptic boutons formed by individual projection neurons. Finally, behavioral analyses suggest that such increased connectivity enhances learning performance in an associative task. Our study shows how fine-grained aspects of connectivity architecture in an associative brain center can change during evolution to reflect the chemical ecology of a species.

Mushroom body input connections form independently of sensory activity in Drosophila melanogaster.

Associative brain centers, such as the insect mushroom body, need to represent sensory information in an efficient manner. In Drosophila melanogaster, the Kenyon cells of the mushroom body integrate inputs from a random set of olfactory projection neurons, but some projection neurons-namely those activated by a few ethologically meaningful odors-connect to Kenyon cells more frequently than others. This biased and random connectivity pattern is conceivably advantageous, as it enables the mushroom body to represent a large number of odors as unique activity patterns while prioritizing the representation of a few specific odors. How this connectivity pattern is established remains largely unknown. Here, we test whether the mechanisms patterning the connections between Kenyon cells and projection neurons depend on sensory activity or whether they are hardwired. We mapped a large number of mushroom body input connections in partially anosmic flies-flies lacking the obligate odorant co-receptor Orco-and in wild-type flies. Statistical analyses of these datasets reveal that the random and biased connectivity pattern observed between Kenyon cells and projection neurons forms normally in the absence of most olfactory sensory activity. This finding supports the idea that even comparatively subtle, population-level patterns of neuronal connectivity can be encoded by fixed genetic programs and are likely to be the result of evolved prioritization of ecologically and ethologically salient stimuli.

Photo-labeling neurons in the Drosophila brain.

Many genetically encoded tools, including large collections of GAL4 transgenic lines, can be used to visualize neurons of the Drosophila melanogaster brain. However, identifying transgenic lines that are expressed sparsely enough to label individual neurons, or groups of neurons that innervate a particular brain region, remains technically challenging. Here, we provide a detailed procedure in which we used broadly expressed transgenic lines and two-photon microscopy to photo-label neurons with specificity, thereby permitting their morphological characterization. For complete details on the use and execution of this protocol, please refer to Li et al. (2020).

Visual Input into the Drosophila melanogaster Mushroom Body.

The patterns of neuronal connectivity underlying multisensory integration, a fundamental property of many brains, remain poorly characterized. The Drosophila melanogaster mushroom body-an associative center-is an ideal system to investigate how different sensory channels converge in higher order brain centers. The neurons connecting the mushroom body to the olfactory system have been described in great detail, but input from other sensory systems remains poorly defined. Here, we use a range of anatomical and genetic techniques to identify two types of input neuron that connect visual processing centers-the lobula and the posterior lateral protocerebrum-to the dorsal accessory calyx of the mushroom body. Together with previous work that described a pathway conveying visual information from the medulla to the ventral accessory calyx of the mushroom body, our study defines a second, parallel pathway that is anatomically poised to convey information from the visual system to the dorsal accessory calyx.