Higher-order olfactory neurons in the lateral horn support odor valence and odor identity coding in Drosophila

Understanding neuronal representations of odor-evoked activities and their progressive transformation from the sensory level to higher brain centers features one of the major aims in olfactory neuroscience. Here, we investigated how odor information is transformed and represented in higher-order neurons of the lateral horn, one of the higher olfactory centers implicated in determining innate behavior, using Drosophila melanogaster. We focused on a subset of third-order glutamatergic lateral horn neurons (LHNs) and characterized their odor coding properties in relation to their presynaptic partner neurons, the projection neurons (PNs) by two-photon functional imaging. We show that odors evoke reproducible, stereotypic, and odor-specific response patterns in LHNs. Notably, odor-evoked responses in these neurons are valence-specific in a way that their response amplitude is positively correlated with innate odor preferences. We postulate that this valence-specific activity is the result of integrating inputs from multiple olfactory channels through second-order neurons. GRASP and micro-lesioning experiments provide evidence that glutamatergic LHNs obtain their major excitatory input from uniglomerular PNs, while they receive an odor-specific inhibition through inhibitory multiglomerular PNs. In summary, our study indicates that odor representations in glutamatergic LHNs encode hedonic valence and odor identity and primarily retain the odor coding properties of second-order neurons.


Sample-size estimation
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Replicates
• You should report how often each experiment was performed • You should include a definition of biological versus technical replication • The data obtained should be provided and sufficient information should be provided to indicate the number of independent biological and/or technical replicates • If you encountered any outliers, you should describe how these were handled • Criteria for exclusion/inclusion of data should be clearly stated • High-throughput sequence data should be uploaded before submission, with a private link for reviewers provided (these are available from both GEO and ArrayExpress) Please outline where this information can be found within the submission (e.g., sections or figure legends), or explain why this information doesn't apply to your submission: This information can be found in the figure legends for functional imaging experiments and in the section 'materials and methods' for behavioral experiments. The sample size used corresponds to the common sample sizes in the field of Drosophila olfaction functional/behavioral analysis. Statistical analyses are mentioned in the figure legends. In general, the 'Past' software was used to identify the appropriate statistical method according to the type of data, data distribution etc. No power analysis was used and replicates were determined according to the standard procedures in the field.
N-values are specified in each figure legend. Briefly, all experiments were independently carried out several times and several experiments were independently repeated in a similar way. No data were excluded.

Statistical reporting
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Please outline where this information can be found within the submission (e.g., sections or figure legends), or explain why this information doesn't apply to your submission: (For large datasets, or papers with a very large number of statistical tests, you may upload a single table file with tests, Ns, etc., with reference to sections in the manuscript.)

Group allocation
• Indicate how samples were allocated into experimental groups (in the case of clinical studies, please specify allocation to treatment method); if randomization was used, please also state if restricted randomization was applied • Indicate if masking was used during group allocation, data collection and/or data analysis Please outline where this information can be found within the submission (e.g., sections or figure legends), or explain why this information doesn't apply to your submission: Additional data files ("source data") • We encourage you to upload relevant additional data files, such as numerical data that are represented as a graph in a figure, or as a summary table • Where provided, these should be in the most useful format, and they can be uploaded as "Source data" files linked to a main figure or table • Include model definition files including the full list of parameters used • Include code used for data analysis (e.g., R, MatLab) • Avoid stating that data files are "available upon request" Please indicate the figures or tables for which source data files have been provided: The information on statistical methods is given in the appropriate figure legends. The figures show mainly raw data distributions within box plots or bar plots. Exact p-values are mentioned in the corresponding figures; in addition p-values are reported using number of stars defined in the figure legends. Analyzing methods were chosen according to sample distribution, variation, size, groups and appropriate tests were determined using the 'Past' software.
For functional imaging experiments, groups were defined by the genotype of the flies. For the data analyses, groups were chosen based on hedonic valence of the odors, which are defined in the figures, figure legends and in the appropriate text. For behavioral experiments with wild type flies, groups were chosen randomly and aged to the appropriate ages (as described in the Methods). We provide all source data of this study. The fluorescent changes of GCaMP6f obtained by 2-photon functional imaging deriving from the experiments that are represented as graphs in the main and supplementary figure are provided as excel files (Figs. 2,3,5,6). All raw data files of the functional imaging experiments (Figs. 2,3,5,6), the behavioral experiments (Fig. 3), the photoactivation and immunohistochemistry experiments (Figs. 1, 4) have been deposited on the Edmond server, the Open Research Data Repository of the Max Planck Society, which can be accessed via the following link once the article is published: