Dorsal raphe nucleus to anterior cingulate cortex 5-HTergic neural circuit modulates consolation and sociability

Consolation is a common response to the distress of others in humans and some social animals, but the neural mechanisms underlying this behavior are not well characterized. By using socially monogamous mandarin voles, we found that optogenetic or chemogenetic inhibition of 5-HTergic neurons in the dorsal raphe nucleus (DR) or optogenetic inhibition of serotonin (5-HT) terminals in the anterior cingulate cortex (ACC) significantly decreased allogrooming time in the consolation test and reduced sociability in the three-chamber test. The release of 5-HT within the ACC and the activity of DR neurons were significantly increased during allogrooming, sniffing, and social approaching. Finally, we found that the activation of 5-HT1A receptors in the ACC was sufficient to reverse consolation and sociability deficits induced by the chemogenetic inhibition of 5-HTergic neurons in the DR. Our study provided the first direct evidence that DR-ACC 5-HTergic neural circuit is implicated in consolation-like behaviors and sociability.

& 2. Optogenetic modulation of 5-HT neuron in the DR and 5-HT terminals in the ACC. N = 7-8 in each group. In the consolation test, independent samples t tests along with Bayesian independent samples t-test; in the open-filed and three-chamber test, two-way repeated measures ANOVA along with two-way repeated Bayesian measures ANOVA (light as within subject factor). The significant level was set at P < 0.05. Figure 3. Chemogenetic modulation of DR 5-HT neuron activities in the DR→ACC neural circuit. N = 7-8 in each group, *P < 0.05, **P < 0.01 compared with vehicle control. For F, one tailed paired t test along with one tailed Bayesian paired samples ttest; for G-I, two-way repeated measures ANOVA along with two-way repeated Bayesian measures ANOVA. Figure 4&5. Fiber photometry recording DR 5-HT neural dynamics and 5-HT release within the ACC during the consolation test. N = 5 in each group; for 'ΔF/F' value comparisons around the behaviors, paired samples t-test along with Bayesian paired samples t-test. Figure 6. Intra-ACC injection of a 5-HT1AR agonist rescued sociability deficits induced by chemogenetic inhibition of DR 5-HT neurons in the DR→ACC neural circuit. N = 5-6 in each group, two-way ANOVA along with two-way Bayesian ANOVA along with Tukey post -hoc test.

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: Statistical reporting  Statistical analysis methods should be described and justified  Raw data should be presented in figures whenever informative to do so (typically when N per group is less than 10)  For each experiment, you should identify the statistical tests used, exact values of N, definitions of center, methods of multiple test correction, and dispersion and precision measures (e.g., mean, median, SD, SEM, confidence intervals; and, for the major substantive results, a measure of effect size (e.g., Pearson's r, Cohen's d)  Report exact p-values wherever possible alongside the summary statistics and 95% confidence intervals. These should be reported for all key questions and not only when the p-value is less than 0.05.
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 Method section.
For Figure 1-3 and Figure 6, behavioral responses of each animal were scored and quantified just once. For Figure 4 and 5, fiber photometry signals were recorded only once unless a specific behavior occurred no more than four times during the 1-h test.
In this case, the record was replicated in the following day.
For all the behavioral performance, some subjects show little or no movements, i.e., they just sited there during the test. We think these individuals did not show normal activity and motivation, therefore not appropriate to include their data in the following analysis. The sample sizes are '7' in some cases. (Source data file; Figure 1I: Male_ChR2 group, Female_ChR2 group; Figure 1L: Male_Dio group; Figure 2H: Male_Dio group; Figure 3G: Male_GI group; Figure 3H: Female_Dio group, Male_Gq_group, Male_GI group; Figure 3I: Female_Dio group, Male_Gq group, Male_GI_group). 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: This information can be found in the figure legends.
Statistical analysis methods, exact values of 'N', the distribution of the raw data had been provided in the figures and figure legends.
Raw data had been presented in the 'source data' document.
The detailed quantifications had been provided in Supplementary 2.

This information can be found in the Method section.
During all the tests, all groups of experimental voles were randomly selected and the experimenters were blinded to the treatments.