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

  1. Laifu Li
  2. Li-Zi Zhang
  3. Zhi-Xiong He
  4. Huan Ma
  5. Yu-Ting Zhang
  6. Yu-Feng Xun
  7. Wei Yuan
  8. Wen-Juan Hou
  9. Yi-Tong Li
  10. Zi-Jian Lv
  11. Rui Jia
  12. Fa-Dao Tai  Is a corresponding author
  1. Institute of Brain and Behavioral Sciences, College of Life Sciences, Shaanxi Normal University, China
  2. College of Life Sciences, Nanyang Normal University, China
  3. Provincial Key Laboratory of Acupuncture and Medications, Shaanxi University of Chinese Medicine, China
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Cite this article as: eLife 2021;10:e67638 doi: 10.7554/eLife.67638

Abstract

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.

Introduction

Consolation behavior, which is referred to as an increase in affiliative contact toward a distressed individual by an uninvolved bystander, is an important component of the social capabilities of humans (de Waal and Preston, 2017; Field et al., 2009). Impaired consolation has been frequently observed in many psychiatric diseases, such as depression, autism, and schizophrenia (Young et al., 2015). According to de Waal’s multilevel conceptualization of empathy (de Waal, 2008; de Waal and Preston, 2017), consolation represents an intermediate level of empathy (the primary level of ‘emotional contagion’, the more complex level of ‘consolation’, and the most elaborate level of ‘perspective taking and targeted helping’), which has long been assumed to exist in species possessing complex cognitive functions, such as humans, apes, dolphins, and elephants (Pérez-Manrique and Gomila, 2018); however, recent studies have indicated that it also exists in some socially lived rodents, such as prairie voles (Burkett et al., 2016), mandarin voles (Li et al., 2019), and rats (Knapska et al., 2010).

Currently, studies of the neural mechanisms underlying consolation and other forms of empathy have primarily focused on the oxytocin systems (Burkett et al., 2016; Li et al., 2019). However, as a complex social behavior, consolation may require the coordinated actions of numerous neuromodulators and neurotransmitters. Serotonin (5-HT) is an evolutionarily ancient neurotransmitter that has long been implicated in a variety of emotional disorders (Faye et al., 2020; Garcia-Garcia et al., 2018; Meneses and Liy-Salmeron, 2012). According to recent studies, 5-HT transmission is also involved in a series of social behaviors such as social interaction (Walsh et al., 2018), social reward, and aggression (Dölen et al., 2013). Regarding empathy, a recent study revealed an association between salivary 5-HT levels and the empathic abilities of people (Matsunaga et al., 2017); a polymorphism in the promoter region of the 5-HT transporter gene has been linked to individual differences in empathy (Gyurak et al., 2013); and MDMA (±3,4 methylenedioxymethamphetamine, better known as the recreational drug ‘ecstasy’), which is well known to stimulate a feeling of closeness and empathy in its users (Carlyle et al., 2019), has been confirmed to robustly increase the release of 5-HT in an activity-independent manner (Heifets and Malenka, 2016). In animal studies, Kim et al., 2014 found that microinjection of 5-HT into the anterior cingulate cortex (ACC) impairs vicarious fear and alters the regularity of neural oscillations in mice. Our recent study has indicated that 5-HT1A receptors within the ACC are involved in consolation deficits induced by chronic social defeat stress in mandarin voles (Li et al., 2020). However, to our knowledge, direct evidence for an association between 5-HT and consolation has yet to be obtained.

Dorsal raphe nucleus (DR) is a main source of 5-HT neurons and provides 70% of 5-HTergic projections in the forebrain (Fu et al., 2010; Luo et al., 2015). DR 5-HTergic neurons form dense, broad, and bidirectional neural connections with a broad range of forebrain and limbic structures, including the ACC (Celada et al., 2013; Charnay and Léger, 2010), which is a central hub for various types of empathy. Therefore, direct modulation of the DR→ACC 5-HTergic circuit to investigate its functional role in consolation-like behaviors is interesting and meaningful.

The released 5-HT binds to pre- and postsynaptic receptors. To date, at least 14 different 5-HT receptor subtypes have been identified in the brain (Artigas, 2013). Among them, 5HT1AR and 5HT2AR are the two main subtypes that are expressed at high levels in the prefrontal cortex (Carhart-Harris and Nutt, 2017; Santana and Artigas, 2017). The distribution, signaling pathways, and functions of these two receptors are substantially different, and both receptors play critical roles in modulating cortical activity and neural oscillations (Celada et al., 2013). Previous studies have indicated that 5HT2AR gene single nucleotide polymorphisms are associated with empathy-related social communication abilities (Gong et al., 2015), and a 5HT2AR agonist increases emotional empathic ability (Dolder et al., 2017). However, in animal studies by Kim et al., 2014, blockade of 5-HT receptors in the ACC did not affect observational fear responses in mice. Clearly, the specific functions of 5HT1AR and 5HT2AR in empathy-like behaviors still require further examination.

The mandarin vole (Microtus mandarinus) is a socially monogamous rodent that is widely distributed across China (He et al., 2019). As shown in our previous studies, this species is capable of displaying consolation-like behaviors upon exposure to a distressed partner (Li et al., 2020; Li et al., 2019). In the present study, we first investigated the function of DR→ACC 5-HTergic circuits in consolation-like behaviors using optogenetic and chemogenetic approaches. To provide more direct evidence, we then monitored ACC 5-HT release and DR neuron activities during this behavior by using in vivo fiber photometry. Finally, we used chemogenetics plus pharmacological approaches to investigate which types of 5-HT receptors in the ACC are involved in consolation-like behaviors in mandarin voles. In order to investigate any potential sex differences during these processes, both male and female subjects were included in our study. As consolation is, in general, a pro-social behavior, some social behaviors were investigated during the tests.

Results

Optogenetic inhibition of DR 5-HT neurons in the DR→ACC neural circuit impaired consolation and reduced sociability

We first determined the 5-HTergic projection relationship between the DR and ACC in mandarin voles. For this experiment, the retrograde tracer cholera toxin subunit B (CTB) was injected into the ACC, followed by immunofluorescence staining of the DR sections with tryptophan hydroxylase 2 (TPH2), a marker of 5-HTergic neurons. A substantial number of TPH2+ neurons colocalized with CTB, indicating the presence of DR-ACC 5-HTergic projections (Figure 1—figure supplement 1). We then used a novel dual-virus optogenetics approach to explore the function of the DR-ACC 5-HTergic circuit in consolation-like behaviors and sociability, where double-floxed AAV-DIO-ChR2-mCherry (DIO-ChR2) or AAV-DIO-eNpHR3.0-mCherry (DIO-eNpHR3.0) was injected into the DR and retro-AAVs containing the TPH2 promoter and Cre element (rAAV(Retro)-TPH2-Cre) were injected into the ACC (Figure 1A). This virus strategy ensures that opsins (excitatory CHR2 or inhibitory eNpHR3.0) are mainly expressed within the DR-ACC 5-HTergic circuit. Immunohistochemical staining showed that more than 75% of mCherry-labeled neurons expressed TPH2 in both male and female voles, and more than 80% of TPH2+ cells coexpressed mCherry (Figure 1—figure supplement 2). In the electrophysiological study, we found that the DR neurons reliably responded to pulses of 473 (activation)/593 (inhibition) nm light stimuli (Figure 1D–E). These results indicate the viability of this virus strategy.

Figure 1 with 5 supplements see all
Optogenetic bidirectional modulation of 5-HT neuron in the DR in the DR-ACC neural circuit.

(A) Schematic of optogenetic manipulation. (B) Timeline of experiments. (C) Immunohistological image showing virus expression in the DR (c1) and amplified images in the left box showing the mCherry, TPH2, and the colocalization of the two (‘c2-c4’). (D) Electrophysiological recording model. (E and F) Representative traces from electrophysiological recordings showing photostimulation (E) and photoinhibition of a 5-HT neuron (F). (G) Quantification of allogrooming time in the consolation test of the CHR2 and control animals (n = 8 in each group; CHR2 vs CTR, independent samples t-test and Bayesian independent samples t-test; male: t(14) = 0.340, p=0.739, BF+0 = 0.445 with median posterior δ = 0.104, 95% CI = [−0.673 to 0.930]; female: t(14) = −0.279, p 0.785, BF+0 = 0.439 with median posterior δ = −0.085, 95% CI = [−0.906 to 0.694]). (H) Quantification of allogrooming time in the consolation test of the eNPHR3.0 and control animals (n = 8 in each group; CHR2 vs eNPHR3.0, independent samples t-test and Bayesian independent samples t-test; male: t(14) = 7.293, p 0.001, BF+0 = 6000.583; female: t(14) = 6.327, p 0.001, BF+0 = 1562.921). (I) Quantification of social preference ratio in the three-chamber test of the CHR2 and control animals (n = 7 in CHR2 groups, one male and one female were excluded from analysis due to immobility; n = 8 in CTR groups; two-way repeated measures ANOVA along with two-way Bayesian repeated measures ANOVA; male: group: F(1, 13) = 3.042, p 0.105, BF(incl) = 1.184; light: F(1, 13) = 0.531, p 0.479, BF(incl) = 0.425; group × light: F(1, 13) = 0.246, p 0.628, BF(incl) = 0.479; female: group: F(1, 13) = 2.088, p 0.172, BF(incl) = 0.790; light: F(1, 13) = 0.180, p 0.678, BF(incl) = 0.426; group × light: F(1, 13) = 0.233, p 0.638, BF(incl) = 0.358). (J) Quantification of social preference ratio in the three-chamber test of the eNPHR3.0 and control animals (n = 8 in each group; two-way repeated measures ANOVA along with two-way Bayesian repeated measures ANOVA; male: group: F(1, 14) = 4.084, p 0.063, BF(incl) = 1.236; light: F(1, 14) = 28.361, p 0.001, BF(incl) = 25.390; group × light: F(1, 14) = 22.959, p 0.001, BF(incl) = 87.850; post-hoc comparisons (Tukey): mCherry_Off vs mCherry_On, p 0.981; eNPHR_Off vs eNPHR_On, p<0.001; female: group: F(1, 14) = 11.892, p 0.004, BF(incl) = 4.965; light: F(1, 14) = 22.678, p 0.001, BF(incl) = 7.067; group × light: F(1, 14) = 33.771, p 0.001, BF(incl) = 623.339; post-hoc comparisons (Tukey): mCherry_Off vs mCherry_On, p 0.879; eNPHR_Off vs eNPHR_On, p 0.001). (K) Quantification of time spent in the central area in the open-field test of the CHR2 and control animals (n = 8 in each group; two-way repeated measures ANOVA along with two-way Bayesian repeated measures ANOVA; male: group: F(1, 14) = 0.009, p 0.465, BF(incl) = 1.184; light: F(1, 14) = 0.808, p 0.384, BF(incl) = 0.442; group × light: F(1, 14) = 2.266, p 0.155, BF(incl) = 0.964; female: group: F(1, 14) = 0.240, p 0.632, BF(incl) = 0.602; light: F(1, 14) = 0.341, p 0.568, BF(incl) = 0.371; group × light: F(1, 14) = 2.192, p 0.161, BF(incl) = 0.910). (L) Quantification of time spent in the central area in the open-field test of the eNPHR3.0 and control animals (male_mCherry, n = 7 (one was excluded from analysis due to immobility); male_eNPHR3.0, n = 8; female_mCherry, n = 8; male_eNPHR3.0, n = 8; two-way repeated measures ANOVA along with two-way Bayesian repeated measures ANOVA; male: group: F(1, 13) = 6.326, p 0.026, BF(incl)=1.935; light: F(1, 13) = 1.176, p 0.298, BF(incl)=0.605; group × light: F(1, 13) = 0.039, p 0.846, BF(incl) = 0.578; post-hoc comparisons (Tukey): mCherry_Off vs mCherry_On, p 0.928; eNPHR_Off vs eNPHR_On, p 0.785; female: group: F(1, 14) = 0.794, p 0.388, BF(incl) = 0.660; light: F(1, 14) = 0.632, p 0.440, BF(incl) = 0.402; group × light: F(1, 14) = 3.390, p 0.087, BF(incl) = 1.352; post-hoc comparisons (Tukey): mCherry_Off vs mCherry_On, p 0.928; eNPHR_Off vs eNPHR_On, p 0.785). Scale bars, 100 μm. Error bars are ± SEM. **p 0.01. For raw data in this figure, please refer to Figure 1—source data 1. ACC: anterior cingulate cortex; Aq: aqueduct; ANOVA: analysis of variance; CTR: control; DR: dorsal raphe nucleus; TPH2: tryptophan hydroxylase 2; 5-HT: serotonin.

Figure 1—source data 1

Source data indicating behavioral performances of bidirectional optogenetic modulation of 5-HT neurons in the DR in the DR-ACC neural circuit.

https://cdn.elifesciences.org/articles/67638/elife-67638-fig1-data1-v2.xls

To test whether modulation of DR 5-HT neuron activity alters consolation and sociability, 5 weeks after the virus injection, optic fibers were implanted above the DR (Figure 1A–B). The virus expression sites and the optic fiber placement schematics are shown in Figure 1—figure supplement 3A. According to Burkett’s and our previous results (Burkett et al., 2016; Li et al., 2019), the time spent grooming their stressed partners (allogrooming) is an important indicator of consolation-like behaviors. We then exposed the subjects to their electric-shocked partners and recorded the time spent allogrooming, chasing (closely following), and selfgrooming (consolation test; for details, please refer to 'Materials and methods'). The three-chamber test was used to assay the sociability along with open-field anxiety and locomotion (Figure 1B). In the CHR2-expressing animals, there was no conclusive evidence that optogenetic activation of 5-HTergic neurons affected their time spent in allogrooming, chasing, and selfgrooming in the 10-min consolation test (Figure 1G, Figure 1—figure supplement 4A–D), the social interaction ratio in the three-chamber test (Figure 1I), and behavioral performance in the open-field test (Figure 1K, Figure 1—figure supplement 4E).

However, in the eNPHR3.0-expressing animals, there was extremely strong evidence that optogenetic inhibition of 5-HT neurons in the DR reduced the time spent allogrooming (Figure 1H) and chasing (Figure 1—figure supplement 4B), but had little effect on the control behavior of selfgrooming (Figure 1—figure supplement 4D). Following experiments indicated that the light-inhibition effects significantly reduced within 24 hr (Figure 1—figure supplement 5). In the three-chamber test, light inhibition also significantly reduced the social preference ratio (Figure 1J). In the open-field test, the results were inconclusive, which suggests that the data for the time spent in the central area and the total distance traveled are equally likely under H0 and H1 (Figure 1L, Figure 1—figure supplement 4F).

The above results indicate that although we have no conclusive results for optogenetic activation, optogenetic inhibition of 5-HT neurons within the DR→ACC 5-HTergic circuit impaired consolation-like behavior and sociability in mandarin voles.

Optogenetic inhibition of ACC 5-HT terminals in the DR→ACC neural circuit similarly impaired consolation and reduced sociability

Modulation of 5-HT neurons may affect other neurons in the DR and thus confound behavioral performance. In subsequent experiments, we placed optic fibers in the ACC and investigated whether the direct modulation of 5-HT terminals in this region would exert the same effects (Figure 2A). Similarly, we found that optogenetic activation of DR-ACC 5-HT terminals did not significantly alter behavioral performance in the consolation test (Figure 2C, Figure 2—figure supplement 1A and C) and open-field test (Figure 2G, Figure 2—figure supplement 1E). In the three-chamber test, two-way repeated measures analysis of variance (ANOVA) revealed a moderate ‘light × group’ interaction in females (F(1, 14) = 6.297, p 0.025, BFincl = 4.186). Subsequent post-hoc comparison results showed that light stimulation slightly increased the social preference ratio in CHR2-expressing females but not in mCherry-expressing females (ChR2: off vs on, p 0.033; mCherry: off vs on, p 0.975; Figure 2E). The ‘light × group’ interaction was not evidently observed in males (F(1, 14) = 3.556, p 0.08, BFincl = 1.239).

Figure 2 with 2 supplements see all
Optogenetic bidirectional modulation of 5-HT terminals within the ACC in the DR-ACC neural circuit.

(A) Schematic of optogenetic manipulation. (B) Timeline of experiments. (C) Quantification of time spent in allogrooming in the consolation test of the CHR2 and control animals (n = 8 in each group; CHR2 vs CTR, independent samples t-test and Bayesian independent samples t-test; male: t(14) = 0.011, p 0.992, BF+0 = 0.428 with median posterior δ = 0.003, 95% CI = [−0.792 to 0.800]; female: t(14) = −0.630, p 0.539, BF+0 = 0.489 with median posterior δ = −0.194, 95% CI = [−1.054 to 0.575]). (D) Quantification of time spent in allogrooming in the consolation test of the eNPHR3.0 and control animals (n = 8 in each group; CHR2 vs CTR, independent samples t-test and Bayesian independent samples t-test; male: t(14) = 8.44, p<0.001, BF+0 = 26556.455; female: t(14) = 12.174, p<0.001, BF+0 = 1.577 × e+6). (E) Quantification of social preference ratio in the three-chamber test of the CHR2 and control animals (n = 8 in each group; two-way repeated measures ANOVA along with two-way Bayesian repeated measures ANOVA; male: group: F(1, 14) = 0.443, p 0.516, BF(incl) = 0.624; light: F(1, 14) = 5.764, p 0.031, BF(incl) = 1.763; group × light: F(1, 14) = 3.556, p 0.080, BF(incl) = 1.239; female: group: F(1, 14) = 3.985, p 0.066, BF(incl) = 1.151; light: F(1, 14) = 3.704, p 0.075, BF(incl) = 1.097; group × light: F(1, 14) = 6.297, p 0.025, BF(incl) = 4.186; post-hoc comparisons (Tukey): mCherry_Off vs mCherry_On, p 0.975; CHR2_Off vs CHR2_On, p 0.033). (F) Quantification of social preference ratio in the three-chamber test of the eNPHR3.0 and control animals (n = 8 in each group; two-way repeated measures ANOVA along with two-way Bayesian repeated measures ANOVA; male: group: F(1, 14) = 9.763, p 0.007, BF(incl) = 3.374; light: F(1, 14) = 13.168, p 0.003, BF(incl)=9.588; group × light: F(1, 14) = 10.843, p 0.005, BF(incl) = 13.892; post-hoc comparisons (Tukey): mCherry_Off vs mCherry_On, p 0.995; eNPHR_Off vs eNPHR_On, p 0.001; female: group: F(1, 14) = 6.912, p 0.020, BF(incl) = 1.545; light: F(1, 14) = 93.330, p 0.001, BF(incl) = 423.687; group × light: F(1, 14) = 65.062, p 0.001, BF(incl) = 20440.982; post-hoc comparisons (Tukey): mCherry_Off vs mCherry_On, p 0.679; eNPHR_Off vs eNPHR_On, p 0.001). (G) Quantification of time spent in the central area in the open-field test of the CHR2 and control animals (n = 8 in each group; two-way repeated measures ANOVA along with two-way Bayesian repeated measures ANOVA; male: group: F(1, 14) = 4.189 × e−4, p 0.984, BF(incl) = 0.483; light: F(1, 14) = 0.842, p 0.374, BF(incl) = 0.489; group × light: F(1, 14) = 0.501, p 0.491, BF(incl) = 0.486; female: group: F(1, 14) = 0.014, p 0.906, BF(incl) = 0.457; light: F(1, 14) = 0.050, p 0.826, BF(incl) = 0.335; group × light: F(1, 14) = 0.177, p 0.681, BF(incl) = 0.440). (H) Quantification of time spent in the central area in the open-field test of the eNPHR3.0 and control animals (male: n = 7, two animals (one from each group) were excluded from analysis due to immobility; female n = 8; two-way repeated measures ANOVA along with two-way Bayesian repeated measures ANOVA; male: group: F(1, 12) = 0.091, p 0.769, BF(incl) = 0.566; light: F(1, 12) = 8.130, p 0.015, BF(incl) = 4.162; group × light: F(1, 12) = 0.802, p 0.388, BF(incl) = 0.550; post-hoc comparisons (Tukey): mCherry_Off vs mCherry_On, p 0.532; eNPHR_Off vs eNPHR_On, p 0.086; female: group: F(1, 14) = 0.276, p 0.607, BF(incl) = 0.578; light: F(1, 14) = 0.580, p 0.459, BF(incl) = 0.404; group × light: F(1, 14) = 2.234, p 0.157, BF(incl) = 0.871). Error bars are ± SEM. *p 0.05, **p 0.01. For raw data in this figure, please refer to Figure 2—source data 1. ACC: anterior cingulate cortex; ANOVA: analysis of variance; DR: dorsal raphe nucleus; CTR: control; 5-HT: serotonin.

Figure 2—source data 1

Source data indicating behavioral performances of optogenetic bidirectional modulation of 5-HT terminals within the ACC in the DR-ACC neural circuit.

https://cdn.elifesciences.org/articles/67638/elife-67638-fig2-data1-v2.xls

Optogenetic inhibition of DR-ACC 5-HT terminals significantly reduced the time spent allogrooming and chasing in the consolation test (Figure 2D, Figure 2—figure supplement 1B) and the social preference ratio in the three-chamber test (Figure 2F). Similarly, the inhibition had little effect on the control behavior of selfgrooming (Figure 2—figure supplement 1D) and behavioral performance in the open-field test (Figure 2H, Figure 2—figure supplement 1F), and the inhibitory effect lasted no more than 24 hr (Figure 2—figure supplement 2). The above results indicated that optogenetic modulation of ACC 5-HT terminals has nearly identical effects as modulation of DR 5-HT neurons.

Chemogenetic inhibition of DR 5-HT neurons in the DR-ACC neural circuit impaired consolation and reduced sociability

To confirm the above optogenetic results over a longer time frame, we used a chemogenetic approach to selectively express ‘Gq-DREADD’ or ‘Gi-DREADD’ in DR 5-HT neurons by injecting AAV-DIO-hM4Dq-mCherry (Gq-DREADD) or AAV-DIO-hM4Di-mCherry (Gi-DREADD) into the DR and rAAV(Retro)-TPH2-Cre into the ACC (Figure 3A). Immunohistochemical staining revealed that more than 65% of TPH2-labeled neurons were infected by mCherry virus and more than 60% of mCherry cells coexpressed TPH2 (Figure 3—figure supplement 1). The virus expression site schematics are shown in Figure 1—figure supplement 3B. To determine whether the ligand clozapine N-oxide (CNO) can activate or inhibit DR 5-HT neurons, whole-cell current-clamp recordings were performed. The results showed that the addition of 10 μM CNO remarkably increased the number of action potentials in the Gq-DREADD-transfected neurons (Figure 3D). In contrast, CNO caused a significant decrease in the number of spikes (Figure 3E) and increased the spike rheobase during current step injections in Gi-DREADD-transfected neurons (Figure 3F). These results indicate the specificity and viability of this virus strategy.

Figure 3 with 2 supplements see all
Chemogenetic modulation of DR 5-HT neuron activities in the DR-ACC neural circuit.

(A) Schematic of chemogenetical manipulations. (B) Timeline of experiments. (C) Immunohistological image showing virus expression in the DR (c1) and amplified images in the left white box showing the mCherry, TPH2, and the colocalization of the two (c2–c4). (D) Representative trace from a Gq-DREADD neuron. (E) Representative trace from a Gi-DREADD-transfected neuron. (F) Quantification of spike rheobase and spike numbers under current step injections in Gi-DREADD-transfected neurons (n = 5 neurons; spike rheobase: paired t-test, t(4) = 4.491, p 0.0109; two-way repeated measures ANOVA; spike numbers: treatment: F(1, 4) = 8.734, p 0.0417, current: F(10, 40) = 8.989, p<0.0001; treatment ×current: F(10, 40) = 4.013, p 0.0008). (G) Quantification of allogrooming time in the consolation test (two-way repeated measures ANOVA along with two-way Bayesian repeated measures ANOVA; male: treatment: F(1, 19) = 6.300, p 0.021, BF(incl) = 2.007; group: F(2, 19) = 5.434, p 0.014, BF(incl) = 0.777; treatment ×group: F(1, 19) = 8.433, p 0.002, BF(incl) = 215.751; post-hoc comparisons (Tukey): Gi_Saline vs Gi_CNO, p 0.002; Gq_Saline vs Gq_CNO, p 1; Dio_Saline vs Dio_CNO, p 0.991). Female: treatment: F(1, 20) = 4.570, p 0.045, BF(incl) = 1.120; group: F(2, 20) = 2.884, p 0.079, BF(incl) = 0.816; treatment × group: F(1, 20) = 10.778, p< 0.001, BF(incl) = 154.421. Post-hoc comparisons (Tukey): Gi_Saline vs Gi_CNO, p 0.001; Gq_Saline vs Gq_CNO, p 0.996; Dio_Saline vs Dio_CNO, p 0.977. (H) Quantification of social preference ratio in the three-chamber test (two-way repeated measures ANOVA along with two-way Bayesian repeated measures ANOVA; male: treatment: F(1, 19) = 4.707, p 0.043, BF(incl) = 2.261; group: F(2, 19) = 1.387, p 0.274, BF(incl) = 0.365; treatment × group: F(1, 19) = 2.990, p 0.074, BF(incl) = 2.562; post-hoc comparisons (Tukey): Gi_Saline vs Gi_CNO, p 0.046; Gq_Saline vs Gq_CNO, p 1; Dio_Saline vs Dio_CNO, p 1; female: treatment: F(1,20) = 11.687, p 0.003, BF(incl) = 9.390; group: F(2, 20) = 0.205, p 0.817, BF(incl) = 0.272; treatment × group: F(1, 20) = 8.923, p 0.002, BF(incl) = 42.856; post-hoc comparisons (Tukey): Gi_Saline vs Gi_CNO, p 0.001; Gq_Saline vs Gq_CNO, p 0.307; Dio_Saline vs Dio_CNO, p 0.892). (I) Quantification of time spent in the central area in the open-field test (two-way repeated measures ANOVA along with two-way Bayesian repeated measures ANOVA; male: treatment: F(1, 19) = 4.305, p 0.052, BF(incl) = 4.438; group: F(2, 19) = 2.064, p 0.155, BF(incl) = 0.384; treatment × group: F(1, 19) = 0.164, p 0.850, BF(incl) = 0.300; post-hoc comparisons (Tukey): Gi_Saline vs Gi_CNO, p 0.963; Gq_Saline vs Gq_CNO, p 0.605; Dio_Saline vs Dio_CNO, p 0.838; female: treatment: F(1, 20) = 0.288, p 0.597, BF(incl) = 0.313; group: F(2, 20) = 4.419, p 0.026, BF(incl) = 2.335; treatment × group: F(1, 20) = 2.242, p 0.132, BF(incl) = 1.146; post-hoc comparisons (Tukey): Gi_Saline vs Gi_CNO, p 0.959; Gq_Saline vs Gq_CNO, p 0.726; Dio_Saline vs Dio_CNO, p 0.711). Male_Dio, n = 8; female_Dio, n = 7; male_Gq, n = 7; female_Gq, n = 8; male_Gi, n = 7; female_Gi, n = 8. Error bars are ± SEM. Scale bars, 100 μm. *p 0.05, **p 0.01. For raw data in this figure, please refer to Figure 3—source data 1. ACC: anterior cingulate cortex; Aq: aqueduct; ANOVA: analysis of variance; DR: dorsal raphe nucleus; TPH2: tryptophan hydroxylase 2; M: male; F: female; 5-HT: serotonin.

Figure 3—source data 1

Source data indicating chemogenetic modulation of DR 5-HT neuron activities in the DR-ACC neural circuit.

https://cdn.elifesciences.org/articles/67638/elife-67638-fig3-data1-v2.xls

In subsequent behavioral studies, we found that CNO (1 mg/kg, intraperitoneally (i.p.))-treated Gi-DREADD-expressing voles showed reduced grooming toward their shocked partners in the consolation test (Figure 3G) and decreased sociability in the three-chamber test (Figure 3H). The CNO-treated males also showed a trend of spending less time in the open-field test, but the statistics were inconclusive (Figure 3I; treatment: F(1,19) = 4.305, p 0.052, BF(incl) = 4.438; group: F(2,19) = 2.064, p 0.155, BF(incl) = 0.384; treatment × group: F(1,19) = 0.164, p 0.850, BF(incl) = 0.300). The treatment had no detectable effects on the total distance traveled in the open-field test (Figure 3—figure supplement 2C) and some control behaviors in the consolation test (Figure 3—figure supplement 2A & B). In Gq-DREADD and virus control subjects, CNO treatment had no detectable effects on the behavioral performance in all the tests (Figure 3).

Based on the results of the chemogenetic and optogenetic experiments described above, we concluded that inhibition of DR-ACC 5-HTergic circuit activity was sufficient to impair consolation and sociability in mandarin voles.

DR 5-HT neuron activity increased during allogrooming and social approaching

If the DR-ACC 5-HTergic circuit is involved in consolation and sociability, ACC projecting DR 5-HT neurons may change their activity during the corresponding behaviors. To verify this idea, we performed photometric recording in freely moving animals by injecting Cre-inducible AAV-DIO-GCaMp6, a genetically encoded fluorescent Ca2+ sensor, into the DR and rAAV(Retro)-TPH2-Cre into the ACC (Figure 4A). 10 days later, fibers were implanted above the DR injection sites. Post-hoc histological analysis revealed that more than 80% GCaMp6+ cells overlapped with TPH2+-expressing cells (Figure 4—figure supplement 1). The virus expression sites and the optic fiber placement schematics are shown in Figure 1—figure supplement 3C.

Figure 4 with 2 supplements see all
Fiber photometry recording DR 5-HT neural dynamics during the consolation test.

(A) Schematic diagrams depicting the virus injection and recording sites. (B) Histology showing the expression of GCaMP6 (left) and GFP control (right) in the DR. (C) Experimental timeline for photometry experiments. (D) Representative fluorescence changes of GCaMP6 (red line) and GFP (blue line) during photometry recordings. (E1–I1) Representative peri-event plot of GCaMP6 fluorescence signals aligned to onsets of various behaviors (for all peri-event plots, the red line denotes the mean signals of four to six bouts of behaviors, whereas the red shaded region denotes the SEM). (E2) Quantification of change in GCaMP6 fluorescence signals before and after allogrooming (n = 6 in each group; male: t(5) = −5.967, p 0.002, BF+0 = 24.488 with median posterior δ = −1.904, 95% CI = [−3.749,–0.424]; female: t(5) = −7.420, p<0.01, BF+0 = 52.689 with median posterior δ = −2.397, 95% CI = [−4.610,–0.625]). (F2) Quantification of change in GCaMP6 fluorescence signals before and after approaching (n = 6 in each group; male: t(5) = −19.871, p<0.001, BF+0 = 2233.691; female: t(5) = −8.448, p 0.001, BF+0 = 84.470 with median posterior δ = −2.747, 95% CI = [−5.225,–0.767]). (G2) Quantification of change in GCaMP6 fluorescence signals before and after sniffing (n = 6 in each group; male: t(5) = −3.689, p 0.011, BF+0 = 6.449 with median posterior δ = −1.221, 95% CI = [−2.576,–0.138]; female: t(5) = −3.689, p 0.014, BF+0 = 5.312 with median posterior δ = −1.137, 95% CI = [−2.434,–0.101]). (H2) Quantification of change in GCaMP6 fluorescence signals before and after selfgrooming (n = 6 in each group; male: t(5) = −1.032, p 0.350, BF+0 = 0.559 with median posterior δ = −0.300, 95% CI = [−1.076, 0.383]; female: t(5) = −0.707, p 0.511, BF+0 = 0.456 with median posterior δ = −0.205, 95% CI = [−0.904, 0.466]). (I2) Quantification of change in GCaMP6 fluorescence signals before and after running (n = 6 in each group; male: t(5) = −1.032, p 0.350, BF+0 = 0.559 with median posterior δ = −0.300, 95% CI = [−1.076, 0.383]; female: t(5) = −0.707, p 0.511, BF+0 = 0.456 with median posterior δ = −0.205, 95% CI = [−0.904, 0.466]). (E3–I3) Representative peri-event plot of GFP signals aligned to onsets of various behavioral events (for all peri-event plots, the blue line denotes the mean signals of four to six bouts of behaviors, whereas the blue shaded region denotes the SE). (E4) Quantification of change in GFP fluorescence signals before and after allogrooming (n = 5 in each group; male: t(4) = −0.145, p 0.885, BF+0 = 0.401 with median posterior δ = −0.047, 95% CI = [−0.3787, 0.675]; female: t(4) = −1.085, p 0.339, BF+0 = 0.610 with median posterior δ = −0.32, 95% CI = [−1.198,–0.406]). (F4) Quantification of change in GFP fluorescence signals before and after approaching (n = 5 in each group; male: t(4) = −1.211, p 0.293, BF+0 = 0.667 with median posterior δ = −0.371, 95% CI = [−1.260, 0.377]; female: t(4) = −0.723, p 0.510, BF+0 = 0.488 with median posterior δ = −0.220, 95% CI = [−1.026, 0.499]). (G4) Quantification of change in GFP fluorescence signals before and after sniffing (n = 5 in each group; male: t(4) = 1.001, p 0.373, BF+0 = 0.577 with median posterior δ = 0.306, 95% CI = [−0.427, 1.156]; female: t(4) = −0.687, p 0.530, BF+0 = 0.479 with median posterior δ = −0.209, 95% CI = [−1.009, 0.510]). (H4) Quantification of change in GFP fluorescence signals before and after selfgrooming (n = 5 in each group; male: t(4) = 0.237, p 0.825, BF+0 = 0.407 with median posterior δ = 0.072, 95% CI = [−0.647, 0.819]; female: t(4) = −0.350, p 0.744, BF+0 = 0.418 with median posterior δ = −0.106, 95% CI = [−0.865, 0.610]). (I4) Quantification of change in GFP fluorescence signals before and after running (n = 5 in each group; paired t-test and Bayesian paired samples t-test, two-tailed; male: t(4) = −0.202, p 0.850, BF+0 = 0.404 with median posterior δ = −0.061, 95% CI = [−0.806, 0.659]; female: t(4) = −1.813, p 0.144, BF+0 = 52.689 with median posterior δ = 0.560, 95% CI = [−1.580,–0.251]). Error bars are ± SEM. Scale bars, 100 μm. *p<0.05, **p<0.01. Paired samples t-test along with Bayesian paired samples t-test. For raw data in this figure, please refer to Figure 4—source data 1. ACC: anterior cingulate cortex; GFP: green fluorescent protein; TPH2: tryptophan hydroxylase 2; Aq: aqueduct; Allo: allogrooming; Sni: sniffing; App: approaching; Gro: selfgrooming; Run: running; 5-HT: serotonin.

Figure 4—source data 1

Source data indicating GCaMP6 and GFP fluorescent signals align to some behaviors.

https://cdn.elifesciences.org/articles/67638/elife-67638-fig4-data1-v2.xls

During the consolation test, we found that the activity of DR 5-HT neurons was reliably increased during allogrooming, social approaching, and sniffing in both sexes (Figure 4E1–E2, F1–F2 and G1–G2). When aligning the fluorescence changes to the end of these social bouts, we found that the activity of 5-HT neurons decreased accordingly before withdrawal from allogrooming and approaching (Figure 4—figure supplement 2A–B). Withdrawing from sniffing also showed a similar trend but was not statistically significant (Figure 4—figure supplement 2C; pre vs post: male: t(5) = 1.611, p 0.084, BF+0 = 1.589 with median posterior δ = 0.528, 95% CI = [0.041, 1.363]; female: t(5) = 1.724, p 0.073, BF+0 = 1.778 with median posterior δ = 0.557, 95% CI = [0.046, 1.415]). The increased activity of DR 5-HT neurons did not simply reflect movement initiation as no fluorescence changes were observed when aligning to running and selfgrooming (Figure 4H1–H2, I1–I2). Furthermore, no significant changes in fluorescence signals were detected in DR 5-HT neurons of green fluorescent protein (GFP)-expressing control voles during all the behaviors, confirming that the GCaMp6 signals genuinely indicated neuronal activity and did not simply reflect motion artifacts (Figure 4E3–E4, F3–F4, G3–G4, H3–H4 and I3–I4).

The above results provide direct evidence that DR 5-HT neurons within the DR-ACC 5-HTergic circuit are recruited in consolation-like behavior and sociability in mandarin voles.

ACC 5-HT release increased during allogrooming, social approach, and sniffing

To further establish the potential relevance of 5-HT in consolation-like behaviors, we directly detected the dynamics of 5-HT within the ACC using a newly developed GPCR-activation-based 5-HT sensor along with photometric recording (Wan et al., 2021). To do this, we injected AAV encoding the fluorescent 5-HT sensor (AAV-5HTsensor 2.1) unilaterally into the ACC and implanted optic fibers above the injection site 10 days later (Figure 5A). The virus expression site and the optic fiber placement schematics are shown in Figure 1—figure supplement 3D. Control animals were injected with AAV encoding enhanced GFP (eGFP). Post-hoc histological verification indicated expression of both the 5-HT sensor and eGFP within the ACC (Figure 5B).

Figure 5 with 2 supplements see all
Fiber photometry recording dynamics of 5-HT within the ACC during the consolation test.

(A) Schematic diagrams depicting the virus injection and recording sites. (B) Histology showing the expression of 5-HT sensor (left) and GFP control (right) within the ACC. (C) Experimental timeline for photometry experiments. (D) Representative fluorescence changes of 5-HT sensor (red line) and GFP (blue line) during photometry recordings. (E1–I1) Representative peri-event plot of 5-HT fluorescence signals aligned to onsets of various behaviors (for all peri-event plots, the red line denotes the mean signals of four to six bouts of behaviors, whereas the red shaded region denotes the SEM). (E2) Quantification of change in 5-HT fluorescence signals before and after allogrooming (n = 6 in each group; male: t(5) = −6.687, p 0.001, BF+0 = 36.393 with median posterior δ = −2.148, 95% CI = [−4.174,–0.523]; female: t(5) = −6.038, p 0.002, BF+0 = 25.495 with median posterior δ = −1.928, 95% CI = [−3.790,–0.433]). (F2) Quantification of change in 5-HT fluorescence signals before and after approaching (n = 6 in each group; male: t(5) = −10.551, p 0.001, BF+0 = 193.396 with median posterior δ = −3.460, 95% CI = [−6.490,–1.060]; female: t(5) = −6.496, p 0.001, BF+0 = 32.865 with median posterior δ = −2.083, 95% CI = [−4.061,–0.497]). (G2) Quantification of change in 5-HT fluorescence signals before and after sniffing (n = 6 in each group; male: t(5) = −5.863, p 0.002, BF+0 = 23.053 with median posterior δ = −1.868, 95% CI = [−3.687,–0.409]; female: t(5) = −3.030, p 0.029, BF+0 = 3.108 with median posterior δ = −0.921, 95% CI = [−2.070, 0.000]). (H2) Quantification of change in 5-HT fluorescence signals before and after selfgrooming (n = 6 in each group; male: t(5) = −0.215, p 0.838, BF+0 = 0.381 with median posterior δ = −0.062, 95% CI = [−0.753, 0.609]; female: t(5) = −0.738, p 0.494, BF+0 = 0.464 with median posterior δ = −0.214, 95% CI = [−0.953, 0.458]). (I2) Quantification of change in 5-HT fluorescence signals before and after running (n = 6 in each group; male: t(5) = −0.627, p 0.558, BF+0 = 0.438 with median posterior δ = −0.182, 95% CI = [−0.908,–0.488]; female: t(5) = 0.129, p 0.903, BF+0 = 0.376 with median posterior δ = 0.037, 95% CI = [−0.636, 0.722]). (E3–I3) Representative peri-event plot of GFP signals aligned to onsets of various behavioral events (for all peri-event plots, the blue line denotes the mean signals of four to six bouts of behaviors, whereas the blue shaded region denotes the SE). (E4) Quantification of change in GFP fluorescence signals before and after allogrooming (n = 5 in each group; male: t(4) = 0.082, p 0.939, BF+0 = 0.399 with median posterior δ = 0.025, 95% CI = [−0.700, 0.760]; female: t(4) = −0.340, p 0.751, BF+0 = 0.610 with median posterior δ = −0.103, 95% CI = [−0.861,–614]). (F4) Quantification of change in GFP fluorescence signals before and after approaching (n = 5 in each group; male: t(4) = −0.836, p 0.450, BF+0 = 0.520 with median posterior δ = −0.255, 95% CI = [−1.078, 0.469]; female: t(4) = 0.861, p 0.438, BF+0 = 0.528 with median posterior δ = 0.263, 95% CI = [−0.462, 1.090]). (G4) Quantification of change in GFP fluorescence signals before and after sniffing (n = 5 in each group; male: t(4) = 2.686, p 0.055, BF+0 = 2.077 with median posterior δ = 0.843, 95% CI = [−0.104, 2.086]; female: t(4) = 0.208, p 0.845, BF+0 = 0.405 with median posterior δ = 0.063, 95% CI = [−0.657, 0.808]). (H4) Quantification of change in GFP fluorescence signals before and after selfgrooming (n = 5 in each group; male: t(4) = 0.822, p 0.457, BF+0 = 0.516 with median posterior δ = 0.251, 95% CI = [−0.473, 1.071]; female: t(4) = 0.653, p 0.549, BF+0 = 0.471 with median posterior δ = 0.199, 95% CI = [−0.519, 0.994]). (I4) Quantification of change in GFP fluorescence signals before and after running (n = 5 in each group; male: t(4) = 1.415, p 0.230, BF+0 = 0.777 with median posterior δ = 0.435, 95% CI = [−0.331, 1.366]; female: t(4) = 0.510, p 0.637, BF+0 = 0.442 with median posterior δ = 0.155, 95% CI = [−0.561, 0.932]). Error bars are ± SEM. Scale bars, 100 μm. *p 0.05, **p 0.01; paired samples t-test along with Bayesian paired samples t-test. For raw data in this figure, please refer to Figure 5—source data 1. ACC: anterior cingulate cortex; M: motor cortex; TPH2: tryptophan hydroxylase 2; Allo: allogrooming; Sni: sniffing; App: approaching; Gro: selfgrooming; Run: running; GFP: green fluorescent protein; 5-HT: serotonin.

Figure 5—source data 1

Source data indicating 5-HT sensor and GFP fluorescent signals align to some behaviors.

https://cdn.elifesciences.org/articles/67638/elife-67638-fig5-data1-v2.xls

Next, we measured the dynamics of endogenous 5-HT during the consolation test. Consistent with the above fiber photometry of Ca2+ signals in DR 5-HT neurons, we found reliably increased fluctuations in fluorescence within the ACC in conjunction with allogrooming, social approaching, and sniffing, but not with selfgrooming and running (Figure 5, left columns). Similarly, the release of 5-HT significantly dropped before the end of allogrooming, approaching, and sniffing (Figure 5—figure supplement 1). In control animals that expressed GFP in the ACC, we observed little fluorescence change during any of these behaviors (Figure 5E3–E4, F3–F4, G3–G4, H3–H4 and I3–I4).

To validate the selectivity of this 5-HT sensor in mandarin voles, in another set of animals expressing the 5-HT sensor, a 5-HT receptor antagonist metergoline (Met) was injected (i.p., 4 mg/kg) before conducting the fiber photometry experiments. We found that treating voles with Met completely blocked the fluorescence changes of the 5-HT sensor during allogrooming (Figure 5—figure supplement 2), validating the viability and specificity of this 5-HT sensors.

The above results provide further evidence that 5-HT released within the ACC may play an important role in consolation-like behaviors and sociability in mandarin voles.

5-HT1A receptors are involved in the reduced consolation and sociability induced by chemogenetic inhibition of DR 5-HT neurons

The most abundant 5-HT receptors expressed in the medial prefrontal cortex (mPFC) are 5-HT1AR and 5-HT2AR (Carhart-Harris and Nutt, 2017). In our previous study, we indicated that ACC administration of a 5HT1AR agonist (8-OH-DPAT) rescued the impaired consolation and sociability induced by chronic social defeat (Li et al., 2020). In the same vein, we used this drug to investigate whether it could relieve the impaired consolation and sociability induced by the inhibition of DR-ACC 5-HTergic circuit, which aimed to clarify the involvement of 5-HT1AR during this process. Considering 5-HT1AR and 5-HT2AR usually exert opposite effects within the mPFC (Celada et al., 2013; Puig and Gulledge, 2011), and 5-HT2A antagonists had been reported to exert anxiolytic effects in preclinical studies (Carson and Kitagawa, 2004), we used MDL 100907 (a 5-HT2A antagonist) here to investigate the possible involvement of 5-HT2AR during this process. To do this, 8-OH-DPAT (200 nl/side; 1.5 mg/ml) or MDL 100907 (200 nl/side; 1 mg/ml) was directly infused into the ACC along with chemogenetic inhibition of DR 5-HT neurons before conducting behavioral assays (Figure 6A and B). Not surprisingly, CNO elicited deficits in allogrooming and sociability (Figure 6C,D,F,G, left two bars). Pretreatment with 8-OH-DPAT significantly reduced the impact of CNO (Figure 6C,D, right two bars; see also the comparison results of ‘Vehicle + Saline - Vehicle + CNO’ vs ‘8-OH-DPAT + Saline - 8-OH-DPAT + CNO’; Figure 6—figure supplement 1A,B), whereas MDL 100907 had no such effect (Figure 6F Figure 6G; right two bars; Figure 6—figure supplement 1C,D). Neither the drugs nor viruses had any detectable effect on the control behavior of selfgrooming or behavioral performance in the open-field test (Figure 6E–H, Figure 6—figure supplement 2).

Figure 6 with 2 supplements see all
Intra-ACC injection of 8-OH-DPAT reduced sociability deficits induced by chemogenetic inhibition of DR 5-HT neurons in the DR→ACC neural circuit.

(A) Schematic representation of ACC infusion sites and virus strategy. (B) Timeline of experimental design. (C) Effect of a 5-HT1AR agonist 8-OH-DPAT on allogrooming time in the consolation test (n = 5 for male, n = 6 for female; male: treatment 1: F(1,16) = 8.230, p 0.011, BF(incl) = 3.005; treatment 2: F(1, 16) = 4.926, p 0.041, BF(incl) = 1.380; treatment 1 × treatment 2: F(1,16) = 4.680, p 0.046, BF(incl) = 1.737; post-hoc comparisons: Veh + Saline vs Veh + CNO, p 0.013; Veh + Saline vs 8-OH-DPAT+CNO, p 0.967; female: treatment 1: F(1, 20) = 5.632, p 0.028, BF(incl) = 1.911; treatment 2: F(1, 20) = 2.746, p 0.113, BF(incl) = 0.831; treatment 1 × treatment 2: F(1, 20) = 4.320, p 0.051, BF(incl) = 1.714; post-hoc comparisons: Veh + Saline vs Veh + CNO, p 0.024; Veh + Saline vs 8-OH-DPAT+CNO, p 0.957). (D) Effect of 8-OH-DPAT on social preference ratio in the three-chamber test (n = 5 for male, n = 6 for female; male: treatment 1: F(1,16) = 12.622, p 0.003, BF(incl) = 3.750; treatment 2: F(1,16) = 4.993, p 0.040, BF(incl) = 0.987; treatment 1 × treatment 2: F(1,16) = 12.622, p 0.003, BF(incl) = 10.906; post-hoc comparisons: Veh + Saline vs Veh + CNO, p 0.001; Veh + Saline vs 8-OH-DPAT+CNO, p 0.788; female: treatment 1: F(1, 20) = 10.820, p 0.004, BF(incl)=6.321; treatment 2: F(1, 20) = 3.075, p 0.095, BF(incl) = 0.858; treatment 1 × treatment 2: F(1, 20) = 5.437, p 0.030, BF(incl) = 2.347; post-hoc comparisons: Veh + Saline vs Veh + CNO, p 0.004; Veh + Saline vs 8-OH-DPAT + CNO, p 0.702). (E) Effect of 8-OH-DPAT on time spent in the central area in the open-field test (n = 5 for male, n = 6 for female; male: treatment 1: F(1,16) = 2.063, p 0.170, BF(incl) = 0.854; treatment 2: F(1,16) = 0.759, p 0.396, BF(incl) = 0.523; treatment 1 × treatment 2: F(1,16) = 0.003, p 0.960, BF(incl) = 0.498; female: treatment 1: F(1, 20) = 0.144, p 0.708, BF(incl) = 0.393; treatment 2: F(1, 20) = 0.324, p 0.575, BF(incl) = 0.422; treatment 1 × treatment 2: F(1, 20) = 0.227, p 0.639, BF(incl) = 0.494). (F) Effect of a 5-HT2AR antagonist (MDL 100907) on allogrooming time in the consolation test (n = 6 in each group; male: treatment 1: F(1, 20) = 37.118, p 0.001, BF(incl) = 1591.039; treatment 2: F(1, 20) = 3.094, p 0.094, BF(incl) = 0.940; treatment 1 × treatment 2: F(1, 20) = 2.697, p 0.116, BF(incl) = 1.101; post-hoc comparisons: Veh + Saline vs Veh + CNO, p 0.001; Veh + Saline vs MDL 100907 + CNO, p<0.001; female: treatment 1: F(1, 20) = 36.168, p<0.001, BF(incl) = 2649.973; treatment 2: F(1, 20) = 0.860, p 0.365, BF(incl) = 0.484; treatment 1 × treatment 2: F(1, 20) = 1.554, p 0.227, BF(incl) = 0.780; post-hoc comparisons: Veh + Saline vs Veh + CNO, p<0.001; Veh + Saline vs MDL 100907 + CNO, p 0.001). (G) Effect of MDL 100907 on social preference ratio in the three-chamber test (n = 6 in each group; male: treatment 1: F(1, 20) = 26.890, p<0.001, BF(incl) = 771.577; treatment 2: F(1, 20) = 0.124, p 0.729, BF(incl) = 0.386; treatment 1 × treatment 2: F(1, 20) = 0.316, p 0.580, BF(incl) = 0.496; post-hoc comparisons: Veh + Saline vs Veh + CNO, p 0.003; Veh + Saline vs MDL 100907 + CNO, p 0.004; female: treatment 1: F(1, 20) = 13.991, p 0.001, BF(incl) = 34.866; treatment 2: F(1, 20) = 0.620, p 0.440, BF(incl) = 0.443; treatment 1 × treatment 2: F(1, 20) = 0.071, p 0.793, BF(incl) = 0.500; post-hoc comparisons: Veh + Saline vs Veh + CNO, p 0.047; Veh + Saline vs MDL 100907 + CNO, p 0.191). (H) Effect of MDL 100907 on time spent in the central area in the open-field test (n = 6 in each group; male: treatment 1: F(1, 20) = 3.702, p 0.069, BF(incl) = 1.469; treatment 2: F(1, 20) = 0.415, p 0.527, BF(incl) = 0.429; treatment 1 × treatment 2: F(1, 20) = 0.270, p 0.609, BF(incl) = 0.520; female: treatment 1: F(1, 20) = 1.506, p 0.234, BF(incl) = 0.655; treatment 2: F(1, 20) = 0.274, p 0.606, BF(incl) = 0.417; treatment 1 × treatment 2: F(1, 20) = 1.305, p 0.267, BF(incl) = 0.698). Two-way ANOVA along with two-way Bayesian ANOVA. Error bars are ± SEM. *p 0.05, **p 0.01. For raw data in this figure, please refer to Figure 6—source data 1. Anta: antangonist; ACC: anterior cingulate cortex; ANOVA: analysis of variance; DR: dorsal raphe nucleus; 5-HT: serotonin.

Figure 6—source data 1

Source data indicating 8-OH-DPAT but not MDL 100907 reduced sociability deficits induced by chemogenetic inhibition of DR 5-HT neurons in the DR→ACC neural circuit.

https://cdn.elifesciences.org/articles/67638/elife-67638-fig6-data1-v2.xls

Discussion

In the present study, we have demonstrated a crucial role for the DR→ACC 5-HTergic neural circuit in the regulation of consolation-like behaviors and sociability in mandarin voles for the first time. Our major findings are listed below. First, inhibition of DR 5-HT neurons or their terminals in the ACC decreased allogrooming behavior and reduced sociability. Second, DR 5-HT neuron activity and ACC 5-HT release increased during allogrooming, social approaching, and sniffing. Third, direct activation of ACC 5-HT1A receptors was sufficient to ameliorate deficits in consolation and sociability induced by chemogenetic inhibition of DR 5-HT neurons.

We found that optogenetic inhibition of DR 5-HT neurons (Figure 1) or ACC 5-HT terminals (Figure 2) significantly decreased intimate behaviors (allogrooming and chasing) toward their distressed partners and reduced sociability. Chemogenetic inhibition of DR 5-HT neurons produced similar results (Figure 3). Consistent with our results, Walsh et al., 2018 found that optogenetic inhibition of DR 5-HT neurons reduced social interactions in the three-chamber test. As consolation is in general a pro-social behavior, the reduced allogrooming may be largely due to an overall decrease in sociability. Further experiments are needed to address this interesting question.

Another open question is how this process occurs, namely, what the neural mechanisms underlying this process are. The neocortical excitatory/inhibitory (E/I) balance hypothesis may help address this question. The hypothesis indicates that an increase in the cortical cellular E/I balance, for example, through increased activity in excitatory neurons or a reduction in inhibitory neuron function, is the common etiology and final pathway for some psychiatric diseases, such as autism and schizophrenia (Bozzi et al., 2018; Vattikuti and Chow, 2010). This hypothesis has recently been verified in mice, as optogenetic excitation of glutamatergic neurons in the mPFC elicits a profound impairment in sociability, while compensatory excitation of inhibitory neurons in this region partially rescues the social deficits caused by an increase in the E/I balance (Yizhar et al., 2011). 5-HT tends to inhibit prefrontal pyramidal activity (Puig and Gulledge, 2011; Tian et al., 2017). For example, according to Puig et al., 2005, electrical stimulation of the DR inhibits approximately two-thirds of pyramidal neurons in the mPFC. Therefore, a reasonable hypothesis is that inhibition of the DR-ACC 5-HTergic neural circuit may increase the E/I balance, which ultimately leads to abnormal social behaviors in mandarin voles. Clearly, this hypothesis should be verified in electrophysiological studies in the future.

In contrast to inhibition manipulations, we found that activation of the DR-ACC 5-HTergic neural circuit did not elicit corresponding increases in allogrooming and sociability (Figures 13). One possible explanation is that there are various 5-HT receptors expressed in the ACC and some have opposite functional effects (Puig and Gulledge, 2011; Tian et al., 2017). For example, 5-HT1AR coupled to the Gi family of G proteins induces hyperpolarization of pyramidal neurons, whereas 5-HT2AR coupled to Gq proteins induces depolarization in the same neurons (Carhart-Harris and Nutt, 2017). In addition, these two types of receptors are expressed in both pyramidal and GABAergic neurons of the mPFC (Santana et al., 2004). Therefore, the net behavioral effects of 5-HT release must result from the combined effects of all these receptors. The other possible explanation is a ceiling effect, which means that the enhancement of consolation and sociability is difficult to accomplish in these normal animals. This may occur in situations where levels of consolation and sociability are low, such as in the stressed subjects in our previous study (Li et al., 2020) or in the genetically less-sociable subjects as in the study by Walsh et al., 2018. However, in a previous study by Kim et al., 2014, the direct administration of 5-HT into the ACC impaired observational fear learning, which is an indicator of emotional contagion. Species differences (mice vs voles), methodological differences (pharmacology vs optogenetics and chemogenetics) or different behavioral indicators (consolation vs emotional contagion) may account for the discrepancies. Furthermore, optogenetic or chemogenetic activation of DR 5-HT neurons is also distinct from pharmacological intervention with MDMA, which robustly induces 5-HT releases in the whole brain and enhances closeness and empathy in its users in human studies (Carlyle et al., 2019; Heifets and Malenka, 2016). Nevertheless, at least some effects of activation were visible. For example, optogenetic activation of ACC 5-HT terminals increased sociability in CHR2-expressing females (Figure 2E). Clearly, the effects of activation of the DR-ACC 5-HTergic neural circuit on sociability and empathy still require further in-depth study.

Our results show that neither activation nor inhibition of the DR-ACC 5-HTergic neural circuit exerts any significant effect on some control behaviors (Figure 1—figure supplement 4; Figure 2—figure supplement 1; Figure 3—figure supplement 2), that is, selfgrooming in the consolation test or behavioral performance in the open-field test, which is consistent with previous results (for selfgrooming, please refer to Correia et al., 2017; for the open-field test, please refer to Walsh et al., 2018). However, inconsistent results have also been reported. For example, Ohmura et al., 2014 found that optogenetic activation of 5-HT neurons in the median raphe nucleus enhanced anxiety-like behaviors in mice. In the study by Correia et al., 2017, activation of 5-HTergic neurons in the DR affected behavior in the open-field test, but not anxiety. Different stimulation protocols or manipulations of different groups of 5-HT neurons may account for these discrepancies.

Our results obtained using in vivo fiber photometry indicated an increase in DR 5-HT neuron activity during allogrooming, sniffing, and social approaching (Figure 4). This result is consistent with previous studies showing increases in the activity of 5-HTergic neurons in the DR during nonaggressive social interactions (Li et al., 2016; Walsh et al., 2018). Furthermore, using the highly sensitive 5-HT fluorescent sensors developed by Wan et al., 2021, we provided the first evidence that allogrooming, sniffing, and social approaching elicit robust 5-HT release in the ACC (Figure 5). Selfgrooming and running did not induce robust 5-HT release within the ACC, which may indicate the behavioral relevance of the 5-HT sensors. We also found that the fluorescence changes in GCamP6 and 5-HT sensors usually dropped before the end of these social behaviors (Figure 4—figure supplement 2, Figure 5—figure supplement 1). These results indicate that 5-HT may be involved in some motivational aspects of these social behaviors, which is consistent with previous findings (Arakawa, 2020; Yagishita, 2020).

Although both 5-HT1AR and 5-HT2AR are expressed at high levels in the mPFC and work together to modulate cortical network activity (Celada et al., 2013), we found only the 5-HT1AR receptor agonist significantly reduced the consolation and sociability deficits induced by chemogenetic inhibition of 5-HTergic neurons in the DR (Figure 6). This result is consistent with our previous finding that the administration of WAY-100635 (a 5-HT1AR antagonist) into the ACC attenuates consolation and sociability in mandarin voles (Li et al., 2020). 5-HT1AR within the mPFC has frequently been reported to exert antidepressant and anxiolytic effects (Artigas, 2013; Fukumoto et al., 2020; Fukumoto et al., 2014; Wang et al., 2019), but our findings clearly indicate that this type of 5-HT receptor within the ACC is also involved in regulating some social behaviors. Clearly, this effect should be verified in many other species in future studies. Administration of the 5-HT2AR antagonist into the ACC did not exert an obvious effect on the social deficits induced by the chemogenetic inhibition of DR 5-HT neurons (Figure 5F–H). Thus, 5-HT2AR within the ACC may not play a major role in consolation and sociability. However, this conclusion should be interpreted very cautiously because 5-HT2AR expression within the mPFC shows clear rostral-to-caudal gradients in mice (Weber and Andrade, 2010) and we were unable to determine its functional role in this dimension in the present study. Furthermore, the effects of 5-HT in the ACC on consolation and sociability probably involve other subtypes of 5-HT receptors, such as 5-HT1BR and 5-HT3R, which requires further examination.

In conclusion, our findings establish the importance of the DR-ACC 5-HTergic neural circuit in consolation-like behaviors and sociability in mandarin voles. One major limitation of our study is that although our virus strategies were generally successful, a few of the infected neurons were indeed TPH2-negative, which may challenge the specificity of our manipulations and confound our results. However, the fiber photometry and pharmacological studies mitigate these concerns. Considering the widespread innervation of 5-HT terminals and abundant distribution of 5-HT receptors in the whole brain, detailed knowledge of cellular and circuit mechanisms of the 5-HTergic system will not only improve our understanding of its complicated features and functions but also have implications for the development of novel therapies for the treatment of prevalent neuropsychiatric disorders, such as depression, autism, and schizophrenia. Additionally, although both male and female subjects were included in this study, sexually dimorphic effects were rarely observed. This finding may provide additional evidence of cooperative evolution to adapt to environmental challenges, particularly in species that adapt to monogamous relationships and require cooperative breeding.

Materials and methods

Key resources table
Reagent type
(species) or
resource
DesignationSource or
reference
IdentifiersAdditional information
AntibodyAnti-TPH2 (goat polyclonal antibody)Abcamab121013;
RRID:AB_10898794
IF (1/500)
AntibodyAlexa Fluor 488-AffiniPure Donkey Anti-Goat IgG (H + L)Jackson705-545-147
RRID:AB_2336933
IF (1/300)
AntibodyRhodamine (TRITC)-AffiniPure Donkey Anti-Goat IgG (H + L)Jackson705-025-003
RRID:AB_2340388
IF (1/300)
Chemical compound, drug8-OH-DPATSigmaH8520Injection site: ACC;
volume: 200 nl/side;
concentration:1.5 mg/ml
Chemical compound, drugMDL 100907Tocris BioscienceCat# 4173Injection site: ACC;
volume:200 nl/side;
concentration:1 mg/ml
Chemical compound, drugMetergolineMedChemExpressCat# HY-B1033/CS-45514 mg/kg, i.p.
Chemical compound, drugCNOBrainVTACat# CNO-021 mg/kg, i.p.
Chemical compound, drugCholera Toxin Subunit B (CTB)594Thermo FisherCat# C34777Injection site: ACC;
volume:400 nl/side;
expression: 10 days
Transfected construct (human)rAAV(Retro)-TPH2-Cre-WPRE-pABrainVTAR-396-K181127Serotype: AAV2;
titer: 5.86 × 1012 vg/ml;
injection site: ACC;
volume: 300 nl/side
Transfected construct (human)rAAV-Ef1α-DIO-ChR2-mCherry-WPRE-pABrainVTA9–2 K190827Serotype: AAV2/9;
titer: 2.01 × 1012 vg/ml;
injection site: DR;
volume: 500 nl
Transfected construct (human)rAAV-Ef1α-DIO-eNpHR3.0-mCherry-WPRE-pABrainVTArAAV9-7-5-1Serotype: AAV2/9;
titer: 2.23 × 1012 vg/ml;
injection site: DR;
volume: 500 nl
Transfected construct (human)rAAV-Ef1α-DIO-mCherry-WPRE-pABrainVTA9–13 K190430Serotype: AAV2/9;
titer: 2.02 × 1012 vg/ml;
injection site: DR;
volume: 500 nl
Transfected construct (human)rAAV-Ef1α-DIO-hM4Di(Gi)-mCherry-WPRE-pABrainVTA9–43 K190521Serotype: AAV2/9;
titer: 2.09 × 1012 vg/ml;
injection site: DR;
volume: 500 nl
Transfected construct (human)rAAV-Ef1α-DIO-hM4Dq(Gq)-mCherry-WPRE-pABrainVTA9–42 K190530Serotype: AAV2/9;
titer: 2.70 × 1012 vg/ml;
injection site: DR;
volume: 500 nl
Transfected construct (human)rAAV-hSyn-5HT2.1-WPRE-hGHpA (5-HT sensor)BrainVTA9–1826 K190620Serotype: AAV2/9;
titer: 2.53 × 1012 vg/ml;
injection site: ACC;
volume: 400 nl
Transfected construct (human)rAAV-hSyn-EGFP-WPRE-hGH-pABrainVTAPT-1990Serotype: AAV2/9;
titer: 2.00 × 1012 vg/ml;
injection site: ACC;
volume: 400 nl
Transfected construct (human)rAAV-EF1α-DIO-GCaMp6-WPRE-hGH pABrainVTAPT-0071Serotype: AAV2/9;
titer: 5.15 × 1012 vg/ml;
injection site: DR;
volume: 500 nl
Transfected construct (human)rAAV-hSyn-DIO-EGFP-WPRE-hGH-PABrainVTA9–1103 K200409Serotype: AAV2/9;
titer: 2.00 × 1012 vg/ml;
injection site: DR;
volume: 500 nl
Software, algorithmJASPJASPRRID:SCR_015823
OtherDAPI stainBosterAR11771 µg/ml

Animals

The mandarin voles used in this study were laboratory-bred strains (F2-F3) whose ancestors were derived from a wild population from Lingbao city (Henan, China). The voles were weaned on postnatal day 21, socially housed in same sex in separate polycarbonate cages (44 × 22 × 16 cm), and housed on a 12 hr light/dark cycle with food and water ad libitum. Voles used for experiments were about 70–90 days old at the time. All breeding, housing, and experimental procedures were in accordance with Chinese guidelines for the care and use of laboratory animals and were approved by the Animal Care and Use Committee of Shaanxi Normal University.

Stereotaxic surgery and virus infusions

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The kind of virus, total injection volumes, and the expression time are listed in Key Resources Table. For surgery, voles (about 50 days) were anesthetized with 1.5–3.0% isoflurane and placed in a stereotaxic instrument. Next, 33-gauge syringe needles (Hamilton) were used for virus delivery. The injection rates were set at 50 nl/min. After each injection, the needle was left in the brain for another 5 min before being slowly withdrawn in order to prevent the virus from leaking out. The bregma coordinates for the virus injection were as follows: ACC: A/P: 1.6, M/L: 0.5, D/V: 1.6; DR: AP: −4.3; DV: −3.3, ML:+1.2, with a 20° angle toward the midline in the coronal plane.

Microinjection and drugs

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The 5-HT1AR agonist 8-OH-DPAT was prepared in saline with a final concentration of 1.5 mg/ml. The 5-HT2AR antagonist MDL 100907 was prepared in 0.01 M phosphate-buffered saline (pH value adjusted to 6.4 with 0.1 M HCl) with a final concentration of 1 mg/ml. All the microinjections were administered 30 min before the behavioral test. The speed of injection was 0.1 μl/min, and the total injection volume was 0.2 µl per side for all the drugs. The injector tips remained in situ for another 2 min for drug diffusions. The dose and timing of drug administration were chosen based on previous studies with 8-OH-DPAT (Cooper et al., 2008; Li et al., 2020) and MDL 100907 (Ishii et al., 2015; Pockros et al., 2011), which were adjusted according to preliminary studies. In chemogenetic studies, CNO (1 mg/kg) was dissolved in saline and delivered i.p. 30 min before the behavioral test.

Optogenetic studies

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For optogenetic activation, ferrules were connected (by patch cords) to a 473 nm laser diode through an FC/PC adaptor and a fiber optic rotary joint. The output parameters were 10 ms, 20 Hz, 8 s on and 2 s off cycle, ∼10 mW for terminal stimulation, ∼5 mW for somatic stimulation. For optogenetic inhibition, ferrules were connected to a 593 nm laser diode. The output parameters were 10 ms, 20 Hz, constant, ∼10 mW for both terminal and somatic inhibition. The optogenetic stimulation parameters were chosen and slightly modified based on previous studies (Garcia-Garcia et al., 2018; Walsh et al., 2018; Zhao et al., 2011).

Behavioral assays

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Generally, all the behavioral experiments were performed under dim light during the dark phase of the light-dark cycle. In all tests, all groups of experimental voles were randomly selected and the observers were blinded to the treatments. During the test, if the subjects (not the stimulus voles) showed very rare or no movements, these individuals were not expected to show normal activity, and their data were not appropriate to include in the following analysis (Figure 1I, L and Figure 2H). For optogenetic and fiber photometry studies, all subjects were allowed to habituate to patch cords for at least 3 days and allowed 30 min acclimation to the connection before the experiment started.

Consolation test

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The consolation test was performed as previously described (Burkett et al., 2016). Briefly, 5 days before the experiment, age-matched adult male and female voles were cohoused together to promote the formation of a pair bond (Yu et al., 2012). On the testing day, the subjects’ partners were gently transferred in a cup to a sound-proof electric shock chamber and then subjected to 10 rounds of moderately strong foot shocks (3 s, 0.8 mA, 2-min intertrial intervals). At the same time, the test voles were left undisturbed in their home cages. After the separation, the pairs were reunited, and the behavior of the subjects was recorded using a video camera for 10 min in the test room. The digital videos were viewed and quantified using J Watcher software (http://www.jwatcher.ucla.edu/). According to previous studies (Burkett et al., 2016; Li et al., 2019), allogrooming is designated as behavioral indicators of consolation, which is defined as head contact with the body or head of their partner, accompanied by a rhythmic head movement. The duration of chasing (following the shocked partners) and selfgrooming was also collected and analyzed.

Optogenetic test was designed as a between-subject test in which both groups (mCherry or opsin) received laser stimulation. The lasting effects of optogenetic inhibition were measured 24 hr after the original test, that is, consolation test was conducted once again without light stimulation in eNPHR 3.0 animals 1 day later.

Three-chamber test

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The three-chamber test was used to assess the sociability of a subject (Horie et al., 2019; Walsh et al., 2018). The apparatus consisted of a rectangular box with three separate chambers (20 × 40 × 20 cm each). One side of each chamber contained a circular metal wire cage (stimulus animal cage, 11 cm high and 9 cm in diameter). 1 day before the test, all subjects were habituated to the arena for 5 min and age- and sex-matched unfamiliar stimulus voles were also habituated to the wire cages for 5 min. On the testing day, the test vole was placed in the center chamber and a ‘stimulus’ vole (housed in the same room with the test voles) was randomly placed into one of the wire cages. After 2 min, the partitioning walls between the chambers were removed and the test voles were allowed to explore freely for a 10 min session. The time spent exploring each chamber was automatically recorded using a video tracking system (Shanghai Xinruan, China). Sociability was calculated as follows: (time spent in the stimulus vole side − time spent in the empty side)/(time spent in the stimulus vole side + time spent in the empty side).

For optogenetic tests, voles received four epochs of light beginning with a light OFF baseline epoch (OFF-ON-OFF-ON). Each epoch lasted for 5 min and the total duration was 20 min.

Open-field test

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The anxiety level and locomotor function of the subjects were assessed by using the open-field test (Flanigan et al., 2020; Walsh et al., 2018). Briefly, a square open field (50 cm × 50 cm × 25 cm) was virtually subdivided into 16 even squares. The four central squares were designated as central area. At the beginning of the test, the test vole was placed into the center area facing away from the experimenter. Behavior was recorded for 5 min. Outcome measures were distance traveled, frequency entries, and time spent in the central area.

For optogenetic tests, voles were tested twice on the same day in both light-off and light-on conditions with at least 2 hr between sessions (within-subject design).

Fiber photometry

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The fiber photometry recording set-up (ThinkerTech, Nanjing, China) was generated and used as previously described (Feng et al., 2019; Yuan et al., 2019). Briefly, the emission light was generated by a 480 LED, reflected with a dichroic mirror, and delivered to the brain in order to excite the GCaMP6m or the 5-HT sensor. The emission light was passed through another band pass filter, into a CMOS detector (Thorlabs, Inc; DCC3240M), and finally recorded by a Labview program (TDMSViewer; ThinkerTech, Nanjing, China).

On the test day, voles were mildly anesthetized with isoflurane and connected to a multi-mode optic fiber patch cord (ThinkerTech Nanjing Bioscience; NA: 0.37, OD: 200 μm) whose other end was connected to a fiber photometry apparatus. After 30-min habituation, the voles were subjected to consolation test as described above (exposure to their shocked partners), and the behavioral recording consisted of allogrooming, sniffing, social approaching, selfgrooming, and running. Four to six bouts of a behavior were collected during the test. If a specific behavior occurred no more than four times within 1 hr, the record was replicated on the following day.

Fiber photometry signals were processed with a custom-written MATLAB software, which is available at https://github.com/ganenlife1980/MultiFiberPhotometry__MetLab (copy archived at swh:1:rev:e89f3a67bb9d40d5e82017f895b2bed74f17a42cThinkerTech Nanjing Bioscience, 2020). Briefly, all the data were segmented based on the behavioral events and baseline phase. The change in fluorescence (ΔF/F) was calculated as (F–F0)/F0, where F0 represents the baseline fluorescence signal averaged over a 10 s-long control time window. We first segmented the data based on the behavioral events. Then, we calculated the average 5-HT and calcium signals in both the pre- and post-phases (2/8 s). The time window was determined according to the duration of a behavior bout. The response elicited during a behavior was calculated as the average ΔF/F during all trials of a particular behavior.

For specificity verification of 5-HT sensors in Figure 5—figure supplement 2, a 5-HT receptor antagonist metergoline (Met) was injected (i.p., 4 mg/kg) 20 min before the fiber photometry experiments. The timing and dosing were chosen according to previous studies (Onasanwo et al., 2016; Wan et al., 2021).

In vitro electrophysiological recordings

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To verify the optogenetic and chemogenetic manipulations, we performed in vitro whole-cell patch-clamp recordings from DR neurons. Neurons expressing ChR2, eNpHR3.0, hM3Dq, and hM4Di were visually identified by fluorescence of mCherry. The voles were anesthetized with isoflurane. Brains were quickly dissected and 300 μm coronal slices containing the DR were prepared in a chamber filled with artificial cerebrospinal fluid (ACSF) (32–34°C) using vibratome (Campden 7000 smz). The recordings were obtained using a Multiclamp 700B amplifier, filtered at 5 kHz, and sampled at 10 kHz with Digidata 1440A. Clampex 10.5 was used for analysis.

Current-clamp recordings were performed to measure evoked action potentials. For photoactivation and photoinhibition, the light protocols used during behavioral tests were delivered through a 200 μm optical fiber close to recorded neurons. For CNO activation, spontaneous firing of action potentials in the cell was recorded in the current-clamp mode at −60 mV. After 5 min of recording, the slices were perfused with 10 μM CNO. The total recording time for each cell was 10 min. For CNO inhibition, we applied currents in steps of 25 pA, ranging from 0 pA to 250 pA. Neurons were allowed to recover for 10 min and then perfused with 10 μM CNO. The same current procedure was performed. Afterwards, the CNO was removed by washes with ASCF and cells were recorded for another 10 min.

Data analysis

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All data are represented as the mean ± SE. All data were assessed for normality using a one-sample Kolmogorov-Smirnov test, and the Levene’s test was used to confirm homogeneity of variance. Comparisons between two groups were performed by unpaired or paired t-tests. Comparisons among three or more groups of different animals were performed using one-way ANOVA. Two-way ANOVAs or two-way repeated measures ANOVAs were used to compare multiple groups under multiple testing conditions when appropriate. Post-hoc comparisons were conducted using Tukey. To confirm the significance and calculate the effect size, Bayesian t-test (paired or unpaired) or Bayesian ANOVA were conducted for all the comparisons using default priors. For more detailed information about the Bayesian test, please refer to Keysers et al., 2020. The data were analyzed using JASP 14.0 and are presented as the mean ± SE. The significant level was set at p 0.05. The detailed analysis method in each figure and the statistical quantifications are presented in Figure 2—figure supplement 2.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files. We have also deposited the datasets of this manuscript into Dryad.

The following data sets were generated
    1. Tai F
    2. Li L
    3. Zhang L-Z
    4. He Z-X
    5. Zhang Y-T
    6. Ma H
    7. Xun Y-F
    8. Yuan W
    9. Hou W-J
    10. Li Y-T
    11. Lv Z-J
    12. Jia R
    (2021) Dryad Digital Repository
    Dorsal raphe nucleus to anterior cingulate cortex 5-HTergic neural circuit modulates consolation and sociability.
    https://doi.org/10.5061/dryad.8931zcrq7

References

    1. Carson WH
    2. Kitagawa H
    (2004)
    Drug development for anxiety disorders: new roles for atypical antipsychotics
    Psychopharmacology Bulletin 38:38–45.
    1. Charnay Y
    2. Léger L
    (2010)
    Brain serotonergic circuitries
    Dialogues in Clinical Neuroscience 12:471–487.
    1. Onasanwo SA
    2. Faborode SO
    3. Ilenre KO
    (2016)
    Antidepressant-like potentials of buchholzia coriacea seed extract: involvement of monoaminergic and cholinergic systems, and neuronal density in the Hippocampus of adult mice
    Nigerian Journal of Physiological Sciences : Official Publication of the Physiological Society of Nigeria 31:93–99.

Decision letter

  1. Kate M Wassum
    Senior Editor; University of California, Los Angeles, United States
  2. Peggy Mason
    Reviewing Editor; University of Chicago, United States
  3. Peggy Mason
    Reviewer; University of Chicago, United States
  4. JP Burkett
    Reviewer; Emory University, United States
  5. Christian Keysers
    Reviewer; Netherlands Institute for Neuroscience, Netherlands

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

While the anterior cingulum is implicated in many affective and social behaviors, this study breaks ground in implicating 5ht dorsal raphe neurons in the same. Given the rich pharmacopeia of drugs acting on 5ht transmission, the findings hold translational promise.

Decision letter after peer review:

[Editors’ note: the authors submitted for reconsideration following the decision after peer review. What follows is the decision letter after the first round of review.]

Thank you for submitting your work entitled "Dorsal raphe nuclei to anterior cingulate cortex 5-HTergic neural circuit modulate consolation and sociability" for consideration by eLife. Your article has been reviewed by 4 peer reviewers, , including Peggy Mason as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen a Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Christian Keysers (Reviewer #2); JP Burkett (Reviewer #3).

This is a multi-pronged study of the contribution of 5ht projections from DR to ACC in consolation behavior in mandarin voles. There was much that the reviewers liked about this study and the manuscript. However, concerns about the photometry data lead us to reject the manuscript, in its present form. There are concerns about the specificity of the method for detecting 5ht. These concerns, well articulated by Reviewer 4 and described below, require new experiments. Should you be able to gather data that assuage these concerns, we would be willing to consider this manuscript as a new submission. While specific reviews are included below, I also highlight here several other concerns that will be critical to address for a successful revision.

– Photometry data: We noted that recordings of the 5HT sensor are confounded by the potential presence of GCAMP in ACC terminals. This could be resolved in several ways for example conducting the 5HT and GCAMP recordings in separate subjects. Validation for the selectivity of the 5HT sensor is needed. A control for motion artifact is also needed.

– Is the observed effect attributable specifically to consolation or could it be a more general effect on sociability? The latter possibility should be considered and discussed.

– The exclusion of data from animals that froze is highly problematic. Please expand on how often this exclusionary criterion was invoked and provide justification for the exclusion.

– The statistical tests used were not compelling to the reviewers. Reviewer #2 outlines many ways to improve this aspect of the presentation. In addition, the authors should consider straightforward equivalence testing, such as equivalence tests (eg TOST) as well as ANOVAs (and MANOVAs) to address correcting for multiple comparisons (see Reviewer 3).

– The change in fluorescence follows the behavior of interest. This is not compatible with the causality implied by the authors' interpretation. This needs to be discussed and possibly aspects of the timing re-analyzed along the lines suggested by Reviewer #2.

– Is consolation behavior at a maximum such that a further enhancement could not be observed with 5ht? The possibility of a ceiling effect as outlined by Reviewer #2 should be considered.

– A schematic showing histological verification of all fiber placements for each experiment is needed. In addition to showing microscopy images showing a larger region of the brain to confirm the image location, it would also help to show a schematic representation of the viral expression spread for each subject (e.g., overlaid on brain slice schematics) for each experiment.

Reviewer #1:

This is a multi-pronged study of the contribution of 5ht projections from DR to ACC in consolation behavior in mandarin voles.

There are writing issues that make less than facile to read. However, these issues can be overcome, and the data are extensive and worthy of consideration despite the challenges to the reader.

The one critical concern that I have is that the authors have not demonstrated that this is consolation behavior as opposed to reunification behavior. In the Burkett et al. study, a very important control of separation without shock was performed. Here that control was not performed. Thus fundamentally the argument that what is being represented here is reunion behavior rather than consolation cannot be refuted by the available evidence.

The behavior should be described in the Results as well as in the methods. eLife is a journal for the general reader who is unlikely to be familiar with the empathy literature.

Are there 5ht terminals in the ACC from median raphe?

Make the scales in Figure 4 constant.

293 Why put a 5ht2ar antagonist while inhibiting DR 5ht neurons? Why not put in an agonist as was done with the 5ht1ar?

Reviewer #2:

In their paper, Li et al., examine the contribution of serotonin in the ACC to consolation behavior in voles. They show that consolation (as measured as an increase of allogroming towards a mate that has been exposed to shocks), as well as social preference (as measured as more time spent close to a conspecific in a 3 compartment test), are reduced when inhibiting the dorsal raphe->anterior cingulate 5HT pathway, and that this can be rescued using a 5HT1A receptor agonist. In addition, they measure transient increases of 5HT in the ACC following the onset of allogrooming and social approach.

In general, this is an interesting and well-conducted study, that combines techniques to interfere with the DR->ACC pathway (including chemo and optogenetic approaches) with some physiology (calcium imaging in DR and 5HT sensors in ACC), to create a series of studies that compel the reader to accept the importance of 5HT on allogroming and social approach.

a) Statistic reporting. The paper contains a large number of data, which is great. However, the presentation of the statistical tests and results is often difficult to understand in detail. I applaud the presence of the data-tables for download, and the rigorous illustration of single subject data in most figures. However, results in the text seldom allow to fully understand what statistical test was used. For instance, in the 3 chamber test in Figure 1, the appropriate test would appear to be a group (mcherry vs Chr2) x condition (laser on vs off) interaction in an ANOVA, which is equivalent to a two-sample t-test of the difference On – Off. Please specify in the text and the figures whether an ANOVA looked at this interaction specifically, and always report exact p-values. Also in the figures, when specific t-test were performed, please indicate the test statistic in the figure (e.g. t(n)=x, p=0.0x), whether significant or not, given there is enough space in the figures. In the current version it is often difficult to assess whether statements of effect or no effect are based on ANOVAs (and if so on what interaction or main effect in that ANOVA) or on posthoc t-tests. I know this makes figures a bit more cluttered, but it provides the reader with a richer understanding of the data.

b) Bayesian statistics. The manuscript contains many highly relevant statements that hinge on the fact that a particular condition had no effect on a particular outcome measure, or that a condition brings contagion levels back to normal. These are statements that frequentist statistics (t-test or ANOVA) cannot support, but that are important in the paper. This is because p>0.05 does not provide evidence for H0 (the absence of an effect). Bayes Factor analyses, in contrast, can easily test (see Keysers, Gazzola and Wagenmakers, Nature Neuroscience, 2020 for details of why and how to do that) and quantify evidence for H0. Therefore please always also provide the Bayes factor for the t-tests, and the Bayesfactor for the inclusion of a factor/interaction in an ANOVA. Given that most of your hypotheses are directional, e.g. inhibition of DR->ACC should reduce allogrooming and social preference, I would recommend using directed hypotheses for the Bayesian test to provide more power in favour of H0. This will indicate whether you have evidence that a manipulation had no effect (BF-0<1/3 or BFincl<1/3) or whether you simply have too much variance or two small a group size to establish that a condition had no effect (1/3<BF<3). These analyses can be easily done using JASP on the tables that you provide, and please indicate the BF-0 values together with the t-values in your figures, and adjust your result description according to whether you have evidence of absence or absence of evidence. I know this is some additional work, and people are only starting to adopt BF testing, but this would really help the reader understand when you really have evidence that a manipulation had no effect (BF<1/3) vs. cases in which your group size is simply too small to exclude an effect (see power considerations in point c).

c) t-test vs ANOVA: in some cases, your statements call for ANOVA's or Bayesian statistics not t-tests. For instance you write: "Pretreatment with 8-OH-DPAT reversed the dysfunctions to the normal level (all P > 0.05 for '8-OH-DPAT+CNO' vs. 'vehicle+saline' pos-hoc comparisons; Figure 5C-D)". Now, to state that it fully reversed to normal level, you need to show that BF-0 for '8-OH-DPAT+CNO' vs. 'vehicle+saline' is <1/3. If you lack power to establish that, you could use an ANOVA to show that 'Vehicle+Saline – Vehicle+CNO > 8-OH-DPAT+Saline – 8-OH-DPAT+CNO', and say that 8-OH-DPAT+CNO significantly reduces the impact of CNO. Both of these would be evidence for something. Currently, simply showing the lack of a significant effect (p>0.05) is simply not evidence for anything, and should be replaced. See Nieuwenhuis, Forstmann and Wagenmakers, (2011) Erroneous Analyses of Interactions in Neuroscience: A Problem of Significance. Nat. Neurosci. 14, 1105-1107, for a more principled discussion of why a lack of significant difference is not the same as a significant interaction. This issue is particularly relevant in this study, that uses small sample sizes as far as statistical power goes, even though I am of course fully aware that larger sample sizes would in so many conditions would not be ethically desirable, but one must be aware that in groups of 8 individuals, and a between group comparison, even large effects (cohen d=0.8) will only be detected in 32% of cases, so even if there is a large effect, you would be more likely not to find it, than to find it, and not finding p<0.05 is thus not evidence for absence even of large effects. After boneferoni correction, things become even worse… This should be carefully addressed throughout the paper, based on ANOVA's or Bayesfactor analyses to qualify statements.

d) Timing of 5HT signals: Line 258+ and Figure 4: it is intriguing that the DeltaF/F seems to increase after onset of the behavior, not prior to onset, if I fully understand the meaning of the '0' point in the Figure 4. The 5HT modulation experiments that you present before seem to suggest causality in the sense of 5HT->social interest and 5HT->allogrooming. For 5HT to trigger those behaviors, one would however expect the fluorescence to precede the behavior, not to follow it. If I understand correctly that the fluorescence follows the behavior rather than preceding them, it would be incompatible with the notion that 5HT triggers the behavior. However 5HT may then maintain the behavior rather than triggering it. Could that be the case? I would suggest providing the following additional data to test this notion: (a) align the fluorescence to the end of the behavior (i.e. moving away from animal in social preference test and stop of grooming or departure from animal in consolation). Would a drop of fluorescence precede the end of the behavior? Second, rather than only plotting the total time in allogrooming or spent with the conspecific, please also present metrics of the duration of the bouts and the number of bouts. If 5HT were to act by prolonging rather than triggering consolation and approach, the bout duration may be a better indicator than the total duration. Please provide this data, and discuss the timing of the fluorescence response and its implications for the role of 5HT in triggering vs. maintaining consolation and approach in the Discussion section.

e) Freezing: Line 482: "if the subject showed evidently immobility, the data were excluded". This statement surprised me, and alerted me to the fact that freezing behavior is not explicitly discussed in the manuscript. Methodologically, I wasn't quite sure what this actually meant: were entire animals removed from the data? How was data-removal performed and how does it affect the presented durations. Does immobility refer to the stressed target of the consolation or the consolator (please specify this throughout the manuscript). Second, freezing has been a key indicator of emotional contagion, and hence of intersubject sensitivity, in the rodent literature. If a stressed demonstrator causes the observer to freeze, this is an important indicator of a reaction to the distress of others, that should really be reported throughout the manuscript. For instance, we know that oxytocin can switch animals between a more active way of coping and a more passive way of coping. If 5HT were to do something like that, it could increase allogrooming, and reduce vicarious freezing in reaction to a stressed demonstrator. Please quantify freezing and immobility, and report it throughout all manipulations, unless it is so rare, that this doesn't make sense. In that case, report briefly how often freezing occurs.

f) Grammar: there are a few grammatical errors in the manuscript. These errors do not compromise the meaning of the manuscript, and are not a big deal, but it may be more elegant to remove them.

Reviewer #3:

This manuscript represents a deep-dive into the role of the raphe-to-cingulate serotonergic circuit in empathy-based consoling behavior in the vole. This is a new and underdeveloped literature, and while this is not the first paper to implicate serotonin in empathy-related behavior in rodents, it is certainly the most methodologically thorough. In addition, the use of vole species allows the authors to measure pro-social behavior in a way not possible in other models.

This research is well designed, well executed, well written, and comprehensively answers the question posed. At every stage, the manuscript anticipated my questions and answered them. It will be well-cited in the field. I strongly recommend it for publication.

1. The authors report several behavioral outcome measures in each behavior test. It would be helpful if the authors listed all of the behavioral outcomes measured in the Methods section for each test. Additionally, they should make clear how statistical corrections for multiple comparisons were conducted, if at all. If no corrections were performed, the authors can simply identify in the Methods which behavior was considered the primary outcome measure and which were secondary/exploratory.

Reviewer #4:

Overall, this is a straightforward paper in which the authors use orthogonal molecular-genetic approaches to determine the necessity of the DRN→ACC pathway in consolation (e.g. allogrooming of a stressed partner) and social investigation. The paper is impressive given the challenges and limitations of implementing many of these techniques in a non-standard model species, such as the mandarin vole. The paper would be strengthened by applying more rigor with respect to interpretation of results and validation of technical approaches.

1) Throughout the paper the authors refer to empathy-like behaviors, as demonstrated by allogrooming in the consolation test. However, there are marked deficits in general sociability from all their manipulations and no other measures of consolation-specific behaviors, making it likely that effects from inhibition of the DRN→ACC on consolation behaviors are not consolation/empathy-specific, but rather a side effect of disruption of sociability or pro-social behaviors in general. Though this issue is addressed briefly in the discussion, it warrants a more consistent acknowledgment and perhaps a reframing of the paper and the overall interpretation of the results. This does not diminish the importance of the author's findings.

2) The application of the dual-site fiber photometry recording, using a novel 5-HT sensor and GCaMP in a non-standard species is impressive. However, there are a number of concerns regarding the implementation of the approach and the specificity of the 5-HT signal. a) There is no demonstration that the 5-HT sensor is specifically providing a readout for extracellular 5-HT. Given that this sensor has not been extensively used/published in vivo (and never in voles), it is important to show that an antagonist blocks the behavior-associated changes in fluorescence (or another relevant validation approach). b) It is possible that the correlation in dF/F observed in the PFC and in the raphe is not due to 5-HT in the PFC but instead due to GCaMP expressed in DRN terminals in the PFC. This would be ideally addressed by imaging in DRN and PFC of GCaMP-only animals. However, it is worth noting that these concerns are mitigated by the pharmacological results in Figure 5. c) There is no explanation for how the authors controlled for motion artifacts, which are concerning given that their analysis focuses on active behaviors. Despite referring to "multi-channel fibre [sic] photometry" in the methods, they describe recording using only a 480 nm LED, so cannot take advantage of an isosbestic signal. Showing that GFP-expressing animals do not show reliable changes in dF/F during these behaviors would ameliorate these concerns.

[Editors’ note: further revisions were suggested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "Dorsal raphe nucleus to anterior cingulate cortex 5-HTergic neural circuit modulates consolation and sociability" for further consideration by eLife. Your revised article has been evaluated by Kate Wassum (Senior Editor) and a Reviewing Editor.

The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:

1) Detail the numbers of animals tested and the number excluded for freezing. It is critical that the reader know the exact number and proportion of animals excluded from each experimental group.

2) The timing of the GCamp signal remains an issue. The timing of which you write: "Interestingly, we found that the fluorescence changes in GCamP6 and 5-HT sensors usually precede allogrooming and social approaching" is not supported by the analysis. See reviewer #3's comments for more detail on this concern.

3) While the title and much of the text along with the results do not distinguish between sociability and consolation, the paragraph on lines 379-388 attempts to make just such a distinction. This should be either changed to bring it in line with the results and remaining text or it should be omitted.

4) Please explain why you would use 8OH-DPAT, an agonist, in the same vein as you used a true 5ht2a antagonist. In the literature, DPAT is often used to reduce 5ht release presumably through effects on presynaptic receptors. But why combine this with the inhibition of 5ht release? Please explain this logic fully. The lines on 339-40 do not explain this sufficiently.

5) In figure 5 E-I it is not clear how the SEM was calculated and it looks as though it is parallel to the average which it would not be if SEM was calculated at each individual time point which is the more appropriate way to convey this information.

6) In the plots to the right of the traces there are only 6 events. It is not clear how many animals were used and which events were used. I think it would be hard to get exactly 6 occurrences of each event.

7) All microscopy images need a scale bar.

8) Please ensure statistical reporting for all main data needs is provided within the main manuscript.

Reviewer #1:

This exciting study demonstrates a requirement for serotonin coming from dorsal raphe and terminating in the anterior cingulate cortex for social behavior including approach and allogrooming associated with consolation. Major strengths include the plethora of techniques brought to bear on a focused question. The only substantial quibble I would have with the authors would be in their efforts to distinguish between consolation and sociability (as measured by approach in a 3-chamber test). They show no distinction and thus whether consolation is primary, secondary or parallel to sociability remains entirely unclear.

I still am unclear on how many animals were excluded for freezing. The authors need to articulate how many animals were tested in each group and the number excluded in each group.

I remain less than convinced that the behavior is consolation rather than sociablity. In their rebuttal, the authors compare allogrooming to self-grooming. This is orthogonal to the point. The key point here is that there are changes in the 3-chamber test that accompany each decrease in consolation behavior; the authors interpret these changes as a decrease in sociability. I do not quibble with the data. I do take exception to the efforts to distinguish between sociability and consolation such as the paragraph on lines 379-388. I do not see grounds for any distinction between sociability and consolation, as the title correctly albeit indirectly suggests.

Other concerns have been allayed.

Reviewer #2:

This is a re-submission of a manuscript I previously reviewed for this journal. The authors have generally answered all of the questions I posed and made all recommended edits. I have no further comments at this time, though I remain concerned about methodological issues previously raised by other reviewers and satisfactory response to these reviewers' concerns is a necessary precondition for publication.

Reviewer #3:

I thank the authors for the thoughtful revision of the paper, and the new data, that further strengthens what is a very strong paper. In particular, the addition of Bayesian statistics, and the careful rephrasing of conclusions in the light of the Bayesian statistics is very helpful. Intuitively, I would have welcomed the inclusion of the key statistics in the figures themselves, rather than in the supplementary 2, but this can be argued either way, and the important factor is that the appropriate statistic is now available and appropriately discussed.

My only remaining comment, which is a nuance rather than a limitation of the paper, regards the photometry signal. In the initial submission, I was surprised that the signal increase followed rather than preceded the consolation behavior. There is now new data, which provides additional evidence that the signal is robustly increased during the social behavior, and together with all the other causal data, this makes a strong case for a relationship between 5HT and these behaviors. The only nuance that I'm still finding difficult to grasp, is the timing relative to onset of the behavior. In the new version you still write: "Interestingly, we found that the fluorescence changes in GCamP6 and 5-HT sensors usually precede allogrooming and social approaching". This is what one would hope for. Unfortunately, I cannot quite see the analysis on which this statement is based. A statistical quantification of the signal in the 100ms before the behavior compared to a further distant baseline might do, or something similar, but currently I really do not see statistical data to support the claim. I would thus suggest either to add a statistical analysis for that, or to remove the statement about this precedence. This is true both in the results and the discussion, where you currently write "Interesting, the increased Ca2+ in DR 5-HT neurons often preceded these behaviors which may indicate the motivational function of 5HT (Arakawa, 2020; Yagishita, 2020)". On the other hand, you now provide nice evidence that it decreases before the end of the behavior, in line with the idea that it maintains and motivates the behavior somehow.

Reviewer #4:

This is a revised manuscript in which the authors address prior reviewer concerns. Specifically, the authors do a good job of addressing concerns regarding the origin of the fluorescent signal in their fiber photometry experiments, clarifying the logic of the paper, employing Bayesian-based statistical approaches, and other improvements. One remaining concern is regarding the logic underlying the pharmacology experiments. Specifically, figure 6 compares data side by side between 5HT1A agonist and 5HT2A antagonist. It is unclear why it was necessary to block the 5HT2A receptor DURING CNO-mediated inhibition of 5-HT release. If the 5HT2A receptor is important how does blocking it when 5-HT is inhibited by CNO test this?

https://doi.org/10.7554/eLife.67638.sa1

Author response

[Editors’ note: the authors resubmitted a revised version of the paper for consideration. What follows is the authors’ response to the first round of review.]

This is a multi-pronged study of the contribution of 5ht projections from DR to ACC in consolation behavior in mandarin voles. There was much that the reviewers liked about this study and the manuscript. However, concerns about the photometry data lead us to reject the manuscript, in its present form. There are concerns about the specificity of the method for detecting 5ht. These concerns, well articulated by Reviewer 4 and described below, require new experiments. Should you be able to gather data that assuage these concerns, we would be willing to consider this manuscript as a new submission. While specific reviews are included below, I also highlight here several other concerns that will be critical to address for a successful revision.

– Photometry data: We noted that recordings of the 5HT sensor are confounded by the potential presence of GCAMP in ACC terminals. This could be resolved in several ways for example conducting the 5HT and GCAMP recordings in separate subjects. Validation for the selectivity of the 5HT sensor is needed. A control for motion artifact is also needed.

Thank you very much for your good suggestions. We conducted the 5HT sensor and GCAMP recordings in separate animals in the revised version (Figure 4, Figure 5). To validate the selectivity of the 5-HT sensor in mandarin voles, a 5-HT receptor antagonist metergoline (Met) was injected (i.p., 4 mg/kg) before conducting the fiber photometry experiments [1,2]. We found the injection of Met completely blocked the fluorescence change of the 5-HT sensor upon allogrooming (Figure 5—figure supplement 2). In control animals that expressed GFP, we observed little changes in fluorescence which excluded the artifacts of motion (Figure 4, Figure 5).

Citations

1. Onasanwo, S.A., Faborode, S.O., Ilenre, K.O., 2016. Antidepressant-like Potentials of Buchholzia Coriacea Seed Extract: Involvement of Monoaminergic and Cholinergic Systems, and Neuronal Density in the Hippocampus of Adult Mice. Nigerian journal of physiological sciences : official publication of the Physiological Society of Nigeria 31, 93-99.

2. Wan JX, Peng WL, Li XL, Qian TG, Song K, Zeng JZ, et al., A genetically encoded GRAB sensor for measuring serotonin dynamics in vivo. Biorxiv. 2020; 38:569-580; posted February 25, 2020; doi: 10.1101/2020.02.24.962282.

–Is the observed effect attributable specifically to consolation or could it be a more general effect on sociability? The latter possibility should be considered and discussed.

Thank you. As consolation is in general a pro-social behavior, the reduced allogrooming is therefore likely due to the overall decrease in sociability. However, we found both DR 5-HT neurons and ACC 5-HT release showed the highest response during allogrooming (Figure 4—figure supplement 2). Both Burkett’s and our previous study indicated that allogrooming toward a distressed partner is a typical feature of consolation [1,2]. Therefore, we think it is appropriate to say that “DRN→ACC 5-HTergic neural circuit play an important regulatory role in consolation and sociability in mandarin voles”. We discussed this point in the revised version (page 21 line 384-388).

“…As consolation is in general a pro-social behavior, it is difficult to determine whether the reduced allogrooming is due to an overall decrease in sociability. However, in the fiber photometry studies, we found both DR 5-HT neurons and ACC 5-HT release showed the highest responses during allogrooming, which may indicate the specific role of 5-HT in consolation-like behaviors (Figure 4—figure supplement 2.)”

Citations

1. Burkett, J. P., Andari, E., Johnson, Z. V., Curry, D. C., de Waal, F. B., and Young, L. J. (2016). Oxytocin-dependent consolation behavior in rodents. Science, 351(6271), 375-378. doi: 10.1126/science.aac4785

2. Li, L. F., Yuan, W., He, Z. X., Wang, L. M., et al., (2019). Involvement of oxytocin and GABA in consolation behavior elicited by socially defeated individuals in mandarin voles. Psychoneuroendocrinology, 103, 14-24. doi: 10.1016/j.psyneuen.2018.12.238

– The exclusion of data from animals that froze is highly problematic. Please expand on how often this exclusionary criterion was invoked and provide justification for the exclusion.

We are very sorry for the confusion. Actually, on rare occasions in the behavioral test, some tested subjects (not the stimulus voles) 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). In the revised version, we had changed the sentence "…if the subject showed evidently immobility, the data were excluded…" into “…During the test, if the subject (not the stimulus voles) showed very rare or no movements, their data were excluded from the following analysis.…”

– The statistical tests used were not compelling to the reviewers. Reviewer #2 outlines many ways to improve this aspect of the presentation. In addition, the authors should consider straightforward equivalence testing, such as equivalence tests (eg TOST) as well as ANOVAs (and MANOVAs) to address correcting for multiple comparisons (see Reviewer 3).

According to reviewer 2’s suggestion, Bayesian test were conducted for all the analysis and ANOVAs were used for multiple comparisons in the revised version. All the statistical quantifications are presented in Supplementary 2.

– The change in fluorescence follows the behavior of interest. This is not compatible with the causality implied by the authors' interpretation. This needs to be discussed and possibly aspects of the timing re-analyzed along the lines suggested by Reviewer #2.

According to your suggestions, we aligned the GCamp6 and 5-HT signal changes to the end of behaviors and found that DeltaF/F values decreased before the end of allogrooming, social approaching and sniffing (Figure 4—figure supplement 3; Figure 5—figure supplement 1). In consideration of DeltaF/F values reliably increased before these behaviors, we think 5-HT may implicate in some motivational aspect of these behaviors. We discussed this point in the revised version (Page 23, Line 450-452).

– Is consolation behavior at a maximum such that a further enhancement could not be observed with 5ht? The possibility of a ceiling effect as outlined by Reviewer #2 should be considered.

We had discussed this point in the revised version (Page 22, Line 413-417).

“…The other possible explanation is a ceiling effect, which means that the enhancement of consolation and sociability are difficult to accomplish in these normal animals. This may occur in situations where levels of consolation and sociability are low such as in the stressed subjects in our previous study (Li et al., 2020) or genetically less sociable subjects as in Walsh’s study (Walsh et al., 2018)”.

– A schematic showing histological verification of all fiber placements for each experiment is needed. In addition to showing microscopy images showing a larger region of the brain to confirm the image location, it would also help to show a schematic representation of the viral expression spread for each subject (e.g., overlaid on brain slice schematics) for each experiment.

A schematic representation of the viral expression and fiber placements for all the experiments were provided in the revised version (Figure 1—figure supplement 3).

We would be happy to transfer the peer reviews, along with the reviewers' names (where they agree), to another journal of your choice. For example, we have recently facilitated the transfer of review material to a range of journals, including Biology Open, BMC Biology, EMBO Reports, eNeuro, Life Science Alliance, the PLOS journals, the Journal of Cell Biology, Journal of Neuroscience, and the Journal of General Physiology. Such transfers are only undertaken in response to an explicit request by the corresponding author. For neuroscience submissions, please note that we participate within the Neuroscience Peer Review Consortium (https://nprc.incf.org/index.php/about/information-for-authors/).

Reviewer #1:

This is a multi-pronged study of the contribution of 5ht projections from DR to ACC in consolation behavior in mandarin voles.

There are writing issues that make less than facile to read. However, these issues can be overcome, and the data are extensive and worthy of consideration despite the challenges to the reader.

We appreciate much for your efforts in handling our manuscript. The revised version had been copyedited by AJE (515B8QJY).

The one critical concern that I have is that the authors have not demonstrated that this is consolation behavior as opposed to reunification behavior. In the Burkett et al. study, a very important control of separation without shock was performed. Here that control was not performed. Thus fundamentally the argument that what is being represented here is reunion behavior rather than consolation cannot be refuted by the available evidence.

Actually, the separation without shock was performed as control in our previous study and we found the allogrooming behavior rarely occurred (Figure 1, Figure 3B) [1]. As in Burkett’s study [2], we found grooming towards their shocked partner is an important behavioral indicator of consolation in socially monogamous mandarin voles.

Citations:

1. Burkett, J. P., Andari, E., Johnson, Z. V., Curry, D. C., de Waal, F. B., and Young, L. J. (2016). Oxytocin-dependent consolation behavior in rodents. Science, 351(6271), 375-378. doi: 10.1126/science.aac4785

2. Li, L. F., Yuan, W., He, Z. X., Wang, L. M., et al., (2019). Involvement of oxytocin and GABA in consolation behavior elicited by socially defeated individuals in mandarin voles. Psychoneuroendocrinology, 103, 14-24. doi: 10.1016/j.psyneuen.2018.12.238

The behavior should be described in the Results as well as in the methods. eLife is a journal for the general reader who is unlikely to be familiar with the empathy literature.

Thank you very much for your suggestions. All the behaviors were referred in both the Results and the Method section in the revised version (Results: page 6, line 120-125; Method: page 30, line 552-556).

Are there 5ht terminals in the ACC from median raphe?

It is well known that the serotonergic neurons are mainly located in the dorsal and median raphe nuclei (DR and MnR, respectively) and both sections have bidirectional connections with the prefrontal cortex [1,2]. But in this study, we injected functional DIO virus (ChR2, eNPHR3.0, Gq, Gi) into the DR. Meanwhile, retro-AAVs containing TPH2 promoter and Cre element (rAAV(Retro)-TPH2-Cre) was injected into the ACC. This dual virus strategy ensures that opsins and DREADD are mainly expressed within the DR-ACC 5-HTergic circuits. In spite of this, we think it is interesting and valuable to further investigate the function role of MnR-ACC 5-HTergic circuits in consolation and sociability in future studies.

Citations

1. Fu, W., Le Maitre, E., Fabre, V., Bernard, J.F., David Xu, Z.Q., Hokfelt, T., 2010. Chemical neuroanatomy of the dorsal raphe nucleus and adjacent structures of the mouse brain. The Journal of comparative neurology 518, 3464-3494.

2. Luo, M., Zhou, J., Liu, Z., 2015. Reward processing by the dorsal raphe nucleus: 5-HT and beyond. Learning and memory (Cold Spring Harbor, N.Y.) 22, 452-460.

Make the scales in Figure 4 constant.

Had corrected.

293 Why put a 5ht2ar antagonist while inhibiting DR 5ht neurons? Why not put in an agonist as was done with the 5ht1ar?

We are very sorry for the confusion. It is well-known that 5HT1AR and 5HT2AR are the two main subtypes that expressed at high levels in the mPFC [1,2]. Both in vitro and in vivo evidences indicate that these two types of receptors exert opposite effects in the mPFC. For example, 5-HT1AR coupled to the Gi family of G proteins induces hyperpolarization of pyramidal neurons, whereas 5-HT2AR coupled to Gq proteins induces depolarization in the same neurons [3]. In our previous study, we had indicated that ACC administration of a 5HT1AR agonist (8-OH-DPAT) rescued the reduced consolation and sociability induced by social defeat [4]. We therefore used a 5-HT2AR antagonist here to investigate if this drug could similarly reverse the chemogenetic inhibition effect. We try to make this point more clearly and easier to understand in the revised version (page 18, Line 339-340).

Citations

1. Carhart-Harris, R. L., and Nutt, D. J. (2017). Serotonin and brain function: a tale of two receptors. J Psychopharmacol, 31(9), 1091-1120.

2. Celada, P., Puig, M. V., and Artigas, F. (2013). Serotonin modulation of cortical neurons and networks. Front Integr Neurosci, 7, 25.

3. Carhart-Harris, R.L., Nutt, D.J., 2017. Serotonin and brain function: a tale of two receptors. Journal of psychopharmacology (Oxford, England) 31, 1091-1120.

4. Li, L. F., Yuan, W., He, Z. X., Ma, H., Xun, Y. F., Meng, L. R.,… Tai, F. D. (2020). Reduced consolation behaviors in physically stressed mandarin voles: involvement of oxytocin, dopamine D2 and serotonin 1A receptors within the anterior cingulate cortex. Int J Neuropsychopharmacol. doi: 10.1093/ijnp/pyz060

Reviewer #2:

In their paper, Li et al., examine the contribution of serotonin in the ACC to consolation behavior in voles. They show that consolation (as measured as an increase of allogroming towards a mate that has been exposed to shocks), as well as social preference (as measured as more time spent close to a conspecific in a 3 compartment test), are reduced when inhibiting the dorsal raphe->anterior cingulate 5HT pathway, and that this can be rescued using a 5HT1A receptor agonist. In addition, they measure transient increases of 5HT in the ACC following the onset of allogrooming and social approach.

In general, this is an interesting and well-conducted study, that combines techniques to interfere with the DR->ACC pathway (including chemo and optogenetic approaches) with some physiology (calcium imaging in DR and 5HT sensors in ACC), to create a series of studies that compel the reader to accept the importance of 5HT on allogroming and social approach.

a) Statistic reporting. The paper contains a large number of data, which is great. However, the presentation of the statistical tests and results is often difficult to understand in detail. I applaud the presence of the data-tables for download, and the rigorous illustration of single subject data in most figures. However, results in the text seldom allow to fully understand what statistical test was used. For instance, in the 3 chamber test in Figure 1, the appropriate test would appear to be a group (mcherry vs Chr2) x condition (laser on vs off) interaction in an ANOVA, which is equivalent to a two-sample t-test of the difference On – Off. Please specify in the text and the figures whether an ANOVA looked at this interaction specifically, and always report exact p-values. Also in the figures, when specific t-test were performed, please indicate the test statistic in the figure (e.g. t(n)=x, p=0.0x), whether significant or not, given there is enough space in the figures. In the current version it is often difficult to assess whether statements of effect or no effect are based on ANOVAs (and if so on what interaction or main effect in that ANOVA) or on posthoc t-tests. I know this makes figures a bit more cluttered, but it provides the reader with a richer understanding of the data.

We appreciate much for your good suggestions. In the revised version, all the analysis methods and statistical quantifications are presented in Supplementary 2.

b) Bayesian statistics. The manuscript contains many highly relevant statements that hinge on the fact that a particular condition had no effect on a particular outcome measure, or that a condition brings contagion levels back to normal. These are statements that frequentist statistics (t-test or ANOVA) cannot support, but that are important in the paper. This is because p>0.05 does not provide evidence for H0 (the absence of an effect). Bayes Factor analyses, in contrast, can easily test (see Keysers, Gazzola and Wagenmakers, Nature Neuroscience, 2020 for details of why and how to do that) and quantify evidence for H0. Therefore please always also provide the Bayes factor for the t-tests, and the Bayesfactor for the inclusion of a factor/interaction in an ANOVA. Given that most of your hypotheses are directional, e.g. inhibition of DR->ACC should reduce allogrooming and social preference, I would recommend using directed hypotheses for the Bayesian test to provide more power in favour of H0. This will indicate whether you have evidence that a manipulation had no effect (BF-0<1/3 or BFincl<1/3) or whether you simply have too much variance or two small a group size to establish that a condition had no effect (1/3<BF<3). These analyses can be easily done using JASP on the tables that you provide, and please indicate the BF-0 values together with the t-values in your figures, and adjust your result description according to whether you have evidence of absence or absence of evidence. I know this is some additional work, and people are only starting to adopt BF testing, but this would really help the reader understand when you really have evidence that a manipulation had no effect (BF<1/3) vs. cases in which your group size is simply too small to exclude an effect (see power considerations in point c).

I must applaud these excellent comments and we learn much for that. It is an amazing experience to conduct Bayesian test with JASP. For clear and concise, all the analysis methods and qualifications are presented in Supplementary 2.

c) t-test vs ANOVA: in some cases, your statements call for ANOVA's or Bayesian statistics not t-tests. For instance you write: "Pretreatment with 8-OH-DPAT reversed the dysfunctions to the normal level (all P > 0.05 for '8-OH-DPAT+CNO' vs. 'vehicle+saline' pos-hoc comparisons; Figure 5C-D)". Now, to state that it fully reversed to normal level, you need to show that BF-0 for '8-OH-DPAT+CNO' vs. 'vehicle+saline' is <1/3. If you lack power to establish that, you could use an ANOVA to show that 'Vehicle+Saline – Vehicle+CNO > 8-OH-DPAT+Saline – 8-OH-DPAT+CNO', and say that 8-OH-DPAT+CNO significantly reduces the impact of CNO. Both of these would be evidence for something. Currently, simply showing the lack of a significant effect (p>0.05) is simply not evidence for anything, and should be replaced. See Nieuwenhuis, Forstmann and Wagenmakers, (2011) Erroneous Analyses of Interactions in Neuroscience: A Problem of Significance. Nat. Neurosci. 14, 1105-1107, for a more principled discussion of why a lack of significant difference is not the same as a significant interaction. This issue is particularly relevant in this study, that uses small sample sizes as far as statistical power goes, even though I am of course fully aware that larger sample sizes would in so many conditions would not be ethically desirable, but one must be aware that in groups of 8 individuals, and a between group comparison, even large effects (cohen d=0.8) will only be detected in 32% of cases, so even if there is a large effect, you would be more likely not to find it, than to find it, and not finding p<0.05 is thus not evidence for absence even of large effects. After boneferoni correction, things become even worse… This should be carefully addressed throughout the paper, based on ANOVA's or Bayesfactor analyses to qualify statements.

According to your suggestions, we reanalyzed the data.

Although we can’t find the BF-0 value for '8-OH-DPAT+CNO' vs. 'vehicle+saline' less than 1/3, we did find 'Vehicle+Saline – Vehicle+CNO > 8-OH-DPAT+Saline – 8-OH-DPAT+CNO' (Figure 6_Figure Supplementary 1A(allogrooming): Male: t(8) = 2.375, P = 0.045, BF+0 = 3.750 with median posterior δ = 0.999, 95%CI = [0.091 to 2.493]; Female: t(10) = 2.639, P = 0.025, BF+0 = 5.482 with median posterior δ = 1.075, 95%CI = [0.123 to 2.453]. Figure 6_ Figure Supplementary 1B (social preference ratio): Male: t(8) = 2.727, P = 0.026, BF+0 = 5.368 with median posterior δ = 1.174, 95%CI = [0.123 to 2.767]; Female: t(10) = 2.273, P = 0.046, BF+0 = 3.609 with median posterior δ = 0.906, 95%CI = [0.087 to 2.203]).

We had changed “…Pretreatment with 8-OH-DPAT reversed the dysfunctions to the normal level” into “…Pretreatment with 8-OH-DPAT significantly reduces the impact of CNO…” in the revised version.

d) Timing of 5HT signals: Line 258+ and Figure 4: it is intriguing that the DeltaF/F seems to increase after onset of the behavior, not prior to onset, if I fully understand the meaning of the '0' point in the Figure 4. The 5HT modulation experiments that you present before seem to suggest causality in the sense of 5HT->social interest and 5HT->allogrooming. For 5HT to trigger those behaviors, one would however expect the fluorescence to precede the behavior, not to follow it. If I understand correctly that the fluorescence follows the behavior rather than preceding them, it would be incompatible with the notion that 5HT triggers the behavior. However 5HT may then maintain the behavior rather than triggering it. Could that be the case? I would suggest providing the following additional data to test this notion: (a) align the fluorescence to the end of the behavior (i.e. moving away from animal in social preference test and stop of grooming or departure from animal in consolation). Would a drop of fluorescence precede the end of the behavior? Second, rather than only plotting the total time in allogrooming or spent with the conspecific, please also present metrics of the duration of the bouts and the number of bouts. If 5HT were to act by prolonging rather than triggering consolation and approach, the bout duration may be a better indicator than the total duration. Please provide this data, and discuss the timing of the fluorescence response and its implications for the role of 5HT in triggering vs. maintaining consolation and approach in the Discussion section.

As suggested, we replicated the GCamp6 and 5-HT fiber photometric experiments in separated animals and found that both the signals reliably increased before allogrooming, social approaching and sniffing (Figure 4 and 5). When aligning the fluorescence signals to the end of these behaviors, the DeltaF/F values invariably decreased before withdrawing from these behaviors (Figure 4—figure supplement 3A-3B; Figure 5—figure supplement 1). All the results indicate that 5HT may be involved in some motivational aspect of these behaviors. This point had been discussed in the revised version (page 14, line 258-259; page 24, line 451-452).

We reviewed our data and found the inhibition manipulations largely impact the number of bouts rather than the bout duration of allogrooming and chasing, etc. This is consistent with the motivational effect of 5-HT. For concise and clear presentation, these data are not presented in the manuscript, but we presented one group of data affiliated with Figure 1H for your reference.

e) Freezing: Line 482: "if the subject showed evidently immobility, the data were excluded". This statement surprised me, and alerted me to the fact that freezing behavior is not explicitly discussed in the manuscript. Methodologically, I wasn't quite sure what this actually meant: were entire animals removed from the data? How was data-removal performed and how does it affect the presented durations. Does immobility refer to the stressed target of the consolation or the consolator (please specify this throughout the manuscript). Second, freezing has been a key indicator of emotional contagion, and hence of intersubject sensitivity, in the rodent literature. If a stressed demonstrator causes the observer to freeze, this is an important indicator of a reaction to the distress of others, that should really be reported throughout the manuscript. For instance, we know that oxytocin can switch animals between a more active way of coping and a more passive way of coping. If 5HT were to do something like that, it could increase allogrooming, and reduce vicarious freezing in reaction to a stressed demonstrator. Please quantify freezing and immobility, and report it throughout all manipulations, unless it is so rare, that this doesn't make sense. In that case, report briefly how often freezing occurs.

We are very sorry for the confusion. Actually, on rare occasions in the behavioral test, some tested subjects (not the stimulus voles) show little or no movements, i.e., they just sited there during all the testing period. We then think it is not appropriate to include these statistics in the following analysis. The sample size is then ‘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). In the revised version, we had changed the sentence "…if the subject showed evidently immobility, the data were excluded…" into “…if the tested subject (not the stimulus voles) showed very little or no movements during the test, the data were excluded from the following analysis…”

For another, we agree with your opinion that freezing is an important emotional indicator in rodents, but it is seldom occurred in our experiments. A paired conditioned stimulus may elicit such response, which just as in Burkett’s study had done. However, we analyzed self-grooming behavior in our experiments which is another important emotional indicator in rodents (Figure 1—figure supplement 4; Figure 2—figure supplement 1; Figure 3—figure supplement 2; Figure 4; Figure 5 and Figure 6—figure supplement 2).

Citations

Burkett, J. P., Andari, E., Johnson, Z. V., Curry, D. C., de Waal, F. B., and Young, L. J. (2016). Oxytocin-dependent consolation behavior in rodents. Science, 351(6271), 375-378. doi: 10.1126/science.aac4785

f) Grammar: there are a few grammatical errors in the manuscript. These errors do not compromise the meaning of the manuscript, and are not a big deal, but it may be more elegant to remove them.

The revised version had been copyedited by AJE (515B8QJY).

Reviewer #3:

This manuscript represents a deep-dive into the role of the raphe-to-cingulate serotonergic circuit in empathy-based consoling behavior in the vole. This is a new and underdeveloped literature, and while this is not the first paper to implicate serotonin in empathy-related behavior in rodents, it is certainly the most methodologically thorough. In addition, the use of vole species allows the authors to measure pro-social behavior in a way not possible in other models.

This research is well designed, well executed, well written, and comprehensively answers the question posed. At every stage, the manuscript anticipated my questions and answered them. It will be well-cited in the field. I strongly recommend it for publication.

We appreciated much for your comments to our manuscript.

1. The authors report several behavioral outcome measures in each behavior test. It would be helpful if the authors listed all of the behavioral outcomes measured in the Methods section for each test. Additionally, they should make clear how statistical corrections for multiple comparisons were conducted, if at all. If no corrections were performed, the authors can simply identify in the Methods which behavior was considered the primary outcome measure and which were secondary/exploratory.

All the analyzed behaviors were referred in both the Results and the Method section in the revised version (Results: page 6, line 122-124; Method: page 30, line 553-556). According to reviewer 2’s suggestions, Bayesian test were conducted for all the analysis and ANOVAs were used for multiple comparisons in the revised version. All analysis methods and the statistical quantifications are presented in Supplementary 2.

Reviewer #4:

Overall, this is a straightforward paper in which the authors use orthogonal molecular-genetic approaches to determine the necessity of the DRN→ACC pathway in consolation (e.g. allogrooming of a stressed partner) and social investigation. The paper is impressive given the challenges and limitations of implementing many of these techniques in a non-standard model species, such as the mandarin vole. The paper would be strengthened by applying more rigor with respect to interpretation of results and validation of technical approaches.

1) Throughout the paper the authors refer to empathy-like behaviors, as demonstrated by allogrooming in the consolation test. However, there are marked deficits in general sociability from all their manipulations and no other measures of consolation-specific behaviors, making it likely that effects from inhibition of the DRN→ACC on consolation behaviors are not consolation/empathy-specific, but rather a side effect of disruption of sociability or pro-social behaviors in general. Though this issue is addressed briefly in the discussion, it warrants a more consistent acknowledgment and perhaps a reframing of the paper and the overall interpretation of the results. This does not diminish the importance of the author's findings.

Thank you very much for your suggestions. Yes, we agree with your viewpoint that the reduced consolation induced by inhibition of the DRN→ACC is likely due to the overall decrease in sociability. However, in the fiber photometry studies, we found both DR 5-HT neurons and ACC 5-HT release showed the largest response during allogrooming (Figure 4—figure supplement 2). Both Burkett’s and our previous study indicated that allogrooming toward a distressed partner is a typical feature of consolation [1,2]. Therefore, we think it is appropriate to say that “DRN→ACC 5-HTergic neural circuit play an important regulatory role in consolation and sociability in mandarin voles”. We discussed this point in the revised version (page 21 line 384-388).

Citations

1. Burkett, J. P., Andari, E., Johnson, Z. V., Curry, D. C., de Waal, F. B., and Young, L. J., (2016). Oxytocin-dependent consolation behavior in rodents. Science, 351(6271), 375-378. doi: 10.1126/science.aac4785

2. Li, L. F., Yuan, W., He, Z. X., Wang, L. M., et al., (2019). Involvement of oxytocin and GABA in consolation behavior elicited by socially defeated individuals in mandarin voles. Psychoneuroendocrinology, 103, 14-24. doi: 10.1016/j.psyneuen.2018.12.238

2) The application of the dual-site fiber photometry recording, using a novel 5-HT sensor and GCaMP in a non-standard species is impressive. However, there are a number of concerns regarding the implementation of the approach and the specificity of the 5-HT signal. a) There is no demonstration that the 5-HT sensor is specifically providing a readout for extracellular 5-HT. Given that this sensor has not been extensively used/published in vivo (and never in voles), it is important to show that an antagonist blocks the behavior-associated changes in fluorescence (or another relevant validation approach). b) It is possible that the correlation in dF/F observed in the PFC and in the raphe is not due to 5-HT in the PFC but instead due to GCaMP expressed in DRN terminals in the PFC. This would be ideally addressed by imaging in DRN and PFC of GCaMP-only animals. However, it is worth noting that these concerns are mitigated by the pharmacological results in Figure 5. c) There is no explanation for how the authors controlled for motion artifacts, which are concerning given that their analysis focuses on active behaviors. Despite referring to "multi-channel fibre [sic] photometry" in the methods, they describe recording using only a 480 nm LED, so cannot take advantage of an isosbestic signal. Showing that GFP-expressing animals do not show reliable changes in dF/F during these behaviors would ameliorate these concerns.

We appreciate much for these excellent comments!According to your suggestions, we conducted the 5HT sensor and GCAMP6 recordings in separate animals in the revised version (Figure 4, Figure 5). Meanwhile, to validate the selectivity of this 5-HT sensor in mandarin voles, a 5-HT receptor antagonist metergoline (Met) was injected (i.p., 4 mg/kg) before conducting fiber photometry recordings. The results showed that Met completely blocked the fluorescence change of the 5-HT sensor along with allogrooming (Figure 5 figure supplement 2). Finally, control animals only expressing GFP were examined in additional experiments (Figure 4, 5), and we found little fluorescence changes along with all the behaviors which excluding the motion

[Editors’ note: what follows is the authors’ response to the second round of review.]

The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:

1) Detail the numbers of animals tested and the number excluded for freezing. It is critical that the reader know the exact number and proportion of animals excluded from each experimental group.

Both the animal numbers and the animals excluded from analysis due to immobility (Figure 1I, Figure 1L and Figure 2H) are listed in the revised figure legends.

2) The timing of the GCamp signal remains an issue. The timing of which you write: "Interestingly, we found that the fluorescence changes in GCamP6 and 5-HT sensors usually precede allogrooming and social approaching" is not supported by the analysis. See reviewer #3's comments for more detail on this concern.

We deleted this statement in the revised version.

3) While the title and much of the text along with the results do not distinguish between sociability and consolation, the paragraph on lines 379-388 attempts to make just such a distinction. This should be either changed to bring it in line with the results and remaining text or it should be omitted.

Admittedly, it is difficult to clarify whether the decrease in sociability is the reason behind the reduced consolation in our study. We therefore try not to distinguish the two in the revised version and just say “…As consolation is in general a pro-social behavior, the reduced allogrooming may be largely due to an overall decrease in sociability. Further experiments are needed to address this interesting question” (page 12, line 279-281).

4) Please explain why you would use 8OH-DPAT, an agonist, in the same vein as you used a true 5ht2a antagonist. In the literature, DPAT is often used to reduce 5ht release presumably through effects on presynaptic receptors. But why combine this with the inhibition of 5ht release? Please explain this logic fully. The lines on 339-40 do not explain this sufficiently.

Actually, 5-HT1A receptors are found in two main populations of neurons: (1) serotonin neurons of the midbrain raphe nuclei (as presynaptic autoreceptors to control the release of 5-HT); and (2) postsynaptic neurons mainly in the hippocampus, septum, amygdala and corticolimbic areas as to modulate neuronal excitability and plasticity.[1] The microinjection of 8OH-DPAT into the ACC may mainly interact with postsynaptic receptors there.

Both 5-HT1AR and 5-HT2AR express abundantly in the mPFC.[2] In our previous study, we found that ACC administration of 8-OH-DPAT rescued the impaired consolation and sociability induced by social defeat.[3] We then used 8-OH-DPAT to investigate whether it could similarly relieve the impaired consolation and sociability induced by inhibition of DR-ACC 5-HTergic circuit, which aimed to clarify the involvement of 5-HT1AR during this process.

Increasing evidences indicate that 5-HT1AR and 5-HT2AR exert opposite effects. For example, 5-HT1AR coupled to the Gi family of G proteins induces hyperpolarization of pyramidal neurons, whereas 5-HT2AR coupled to Gq proteins induces depolarization in the same neurons.[4] Preclinical studies had indicated that 5-HT2A antagonists exerted anxiolytic effects.[5] Therefore, we used MDL 100907 (a 5-HT2A antagonist) here to investigate the possible involvement of 5-HT2AR during this process. We rephrase the sentence in the revised version to make it more logical in the revised version (Page 11, line 246-255).

“…The most abundant 5-HT receptors expressed in the mPFC are 5-HT1AR and 5-HT2AR [4]. In our previous study, we indicated that ACC administration of a 5HT1AR agonist (8-OH-DPAT) rescued the impaired consolation and sociability induced by chronic social defeat [3]. In the same vein, we used this drug to investigate whether it could relieve the impaired consolation and sociability induced by the inhibition of DR-ACC 5-HTergic circuit, which aimed to clarify the involvement of 5-HT1AR during this process. Considering 5-HT1AR and 5-HT2AR usually exert opposite effects within the mPFC [2, 6], and 5-HT2A antagonists had been reported to exert anxiolytic effects in preclinical studies, we then used MDL 100907 (a 5-HT2A antagonist) to investigate the possible involvement of 5-HT2AR during this process…”

Citations

1. Artigas, F., Serotonin receptors involved in antidepressant effects. Pharmacol Ther, 2013. 137(1): p. 119-31.

2. Celada, P., M.V. Puig, and F. Artigas, Serotonin modulation of cortical neurons and networks. Front Integr Neurosci, 2013. 7: p. 25.

3. Li, L.F., et al., Reduced Consolation Behaviors in Physically Stressed Mandarin Voles: Involvement of Oxytocin, Dopamine D2, and Serotonin 1A Receptors Within the Anterior Cingulate Cortex. Int J Neuropsychopharmacol, 2020. 23(8): p. 511-523.

4. Carhart-Harris, R.L. and D.J. Nutt, Serotonin and brain function: a tale of two receptors. J Psychopharmacol, 2017. 31(9): p. 1091-1120.

5. Carson, W.H. and H. Kitagawa, Drug development for anxiety disorders: new roles for atypical antipsychotics. Psychopharmacol Bull, 2004. 38(1): p. 38-45.

5. Puig, M.V. and A.T. Gulledge, Serotonin and prefrontal cortex function: neurons, networks, and circuits. Mol Neurobiol, 2011. 44(3): p. 449-64.

5) In figure 5 E-I it is not clear how the SEM was calculated and it looks as though it is parallel to the average which it would not be if SEM was calculated at each individual time point which is the more appropriate way to convey this information.

The representative images of fluorescence signals in Figure 4 and 5 are exported from a custom-written MATLAB software package developed by ThinkerTech (Nanjing, China). The original source code file could be found in GitHub,

(https://github.com/ganenlife1980/MultiFiberPhotometry__MetLab).

We asked an engineer from this company and he replied that the shades in the images are ‘SEM’ of the signals which reflecting the range and trend of variations. Similar presentations could be found in Wei’s (Figure 1d),[1] Seo’s B (Figure 2e), [2] Yuan’s (Figure 1G),[3] Tang’s (Figure 2I, J)[4] and Flanigan’s[5] (Figures 1 and 3) paper.

Citations:

1. Wei, Y.C., et al., Medial preoptic area in mice is capable of mediating sexually dimorphic behaviors regardless of gender. Nat Commun, 2018. 9(1): p. 279.

2. Seo, C., et al., Intense threat switches dorsal raphe serotonin neurons to a paradoxical operational mode. Science, 2019. 363(6426): p. 538-542.

3. Yuan, Y., et al., Reward Inhibits Paraventricular CRH Neurons to Relieve Stress. Curr Biol, 2019. 29(7): p. 1243-1251.e4.

4. Tang, Y., et al., Social touch promotes interfemale communication via activation of parvocellular oxytocin neurons. Nat Neurosci, 2020. 23(9): p. 1125-1137.

5. Flanigan, M.E., et al., Orexin signaling in GABAergic lateral habenula neurons modulates aggressive behavior in male mice. Nat Neurosci, 2020. 23(5): p. 638-650.

6) In the plots to the right of the traces there are only 6 events. It is not clear how many animals were used and which events were used. I think it would be hard to get exactly 6 occurrences of each event.

We are sorry for the confusion. During the test, the signals of 4-6 bouts of a specific behavior of an animal were collected. The averaged bouts data are then served as the representative value for this behavior of an individual. The details and animal numbers are indicated in the figure legends of the revised version.

7) All microscopy images need a scale bar.

Had added as request.

8) Please ensure statistical reporting for all main data needs is provided within the main manuscript.

All the statistical quantifications are presented in the figure legends in the revised version.

Reviewer #1:

This exciting study demonstrates a requirement for serotonin coming from dorsal raphe and terminating in the anterior cingulate cortex for social behavior including approach and allogrooming associated with consolation. Major strengths include the plethora of techniques brought to bear on a focused question. The only substantial quibble I would have with the authors would be in their efforts to distinguish between consolation and sociability (as measured by approach in a 3-chamber test). They show no distinction and thus whether consolation is primary, secondary or parallel to sociability remains entirely unclear.

Admittedly, it is difficult to clarify whether the decrease in sociability is the reasons behind the reduced consolation in our study. We try not to distinguish the two in the revised version and just say “…As consolation is in general a pro-social behavior, the reduced allogrooming may be largely due to an overall decrease in sociability. Further experiments are needed to address this interesting question” (page 12, line 279-281).

I still am unclear on how many animals were excluded for freezing. The authors need to articulate how many animals were tested in each group and the number excluded in each group.

Both the animal numbers and the animals excluded from analysis (Figure 1I, Figure 1L and Figure 2H) are listed in the revised figure legends.

I remain less than convinced that the behavior is consolation rather than sociablity. In their rebuttal, the authors compare allogrooming to self-grooming. This is orthogonal to the point. The key point here is that there are changes in the 3-chamber test that accompany each decrease in consolation behavior; the authors interpret these changes as a decrease in sociability. I do not quibble with the data. I do take exception to the efforts to distinguish between sociability and consolation such as the paragraph on lines 379-388. I do not see grounds for any distinction between sociability and consolation, as the title correctly albeit indirectly suggests.

Admittedly, it is difficult to clarify whether the decrease in sociability is the reasons behind the reduced consolation in our study. We try not to distinguish the two in the revised version and just say “…As consolation is in general a pro-social behavior, the reduced allogrooming here may be largely due to an overall decrease in sociability. Further experiments are needed to address this interesting question” (page 12, line 279-281).

Reviewer #3:

I thank the authors for the thoughtful revision of the paper, and the new data, that further strengthens what is a very strong paper. In particular, the addition of Bayesian statistics, and the careful rephrasing of conclusions in the light of the Bayesian statistics is very helpful. Intuitively, I would have welcome the inclusion of the key statistics in the figures themselves, rather than in the supplementary 2, but this can be argued either way, and the important factor is that the appropriate statistic is now available and appropriately discussed.

My only remaining comment, which is a nuance rather than a limitation of the paper, regards the photometry signal. In the initial submission, I was surprised that the signal increase followed rather than preceded the consolation behavior. There is now new data, which provides additional evidence that the signal is robustly increased during the social behavior, and together with all the other causal data, this makes a strong case for a relationship between 5HT and these behaviors. The only nuance that I'm still finding difficult to grasp, is the timing relative to onset of the behavior. In the new version you still write: "Interestingly, we found that the fluorescence changes in GCamP6 and 5-HT sensors usually precede allogrooming and social approaching". This is what one would hope for. Unfortunately, I cannot quite see the analysis on which this statement is based. A statistical quantification of the signal in the 100ms before the behavior compared to a further distant baseline might do, or something similar, but currently I really do not see statistical data to support the claim. I would thus suggest either to add a statistical analysis for that, or to remove the statement about this precedence. This is true both in the results and the discussion, where you currently write "Interesting, the increased Ca2+ in DR 5-HT neurons often preceded these behaviors which may indicate the motivational function of 5HT (Arakawa, 2020; Yagishita, 2020)". On the other hand, you now provide nice evidence that it decreases before the end of the behavior, in line with the idea that it maintains and motivates the behavior somehow.

Firstly, we appreciate much for your efforts in helping improve our manuscript. In the revised version, we removed this statement.

Reviewer #4:

This is a revised manuscript in which the authors address prior reviewer concerns. Specifically, the authors do a good job of addressing concerns regarding the origin of the fluorescent signal in their fiber photometry experiments, clarifying the logic of the paper, employing Bayesian-based statistical approaches, and other improvements. One remaining concern is regarding the logic underlying the pharmacology experiments. Specifically, figure 6 compares data side by side between 5HT1A agonist and 5HT2A antagonist. It is unclear why it was necessary to block the 5HT2A receptor DURING CNO-mediated inhibition of 5-HT release. If the 5HT2A receptor is important how does blocking it when 5-HT is inhibited by CNO test this?

It is well-known that 5-HT1AR and 5-HT2AR express abundantly in the mPFC.[1] In our previous study, we found that administration of 8-OH-DPAT in the ACC rescued the impaired consolation and sociability induced by social defeat.[2] We then used 8-OH-DPAT here to investigate whether it could similarly relieve the impaired consolation and sociability induced by inhibition of DR-ACC 5-HTergic circuit, which aimed to clarify the involvement of 5-HT1AR during this process.

Increasing evidences indicate that 5-HT1AR and 5-HT2AR exert opposite effects. For example, 5-HT1AR coupled to the Gi family of G proteins induces hyperpolarization of pyramidal neurons, whereas 5-HT2AR coupled to Gq proteins induces depolarization in the same neurons.[3] Preclinical studies had indicated that 5-HT2A antagonists exerted anxiolytic effects.[4] Therefore, we used MDL 100907 (a 5-HT2A antagonist) here to investigate the involvement of 5-HT2AR during this process. We rephrase the sentence in the revised version to make it more easier to understand in the revised version (Page 11, line 246-255).

“…The most abundant 5-HT receptors expressed in the mPFC are 5-HT1AR and 5-HT2AR [3]. In our previous study, we indicated that ACC administration of a 5HT1AR agonist (8-OH-DPAT) rescued the impaired consolation and sociability induced by chronic social defeat [2]. In the same vein, we used this drug to investigate whether it could relieve the impaired consolation and sociability induced by the inhibition of DR-ACC 5-HTergic circuit, which aiming to clarify the involvement of 5-HT1AR during this process. Considering 5-HT1AR and 5-HT2AR usually exert opposite effects within the mPFC [1, 5], and 5-HT2A antagonists had been reported to exert anxiolytic effects in preclinical studies, we then used MDL 100907 (a 5-HT2A antagonist) to investigate the possible involvement of 5-HT2AR during this process…”

Citations

1. Celada, P., M.V. Puig, and F. Artigas, Serotonin modulation of cortical neurons and networks. Front Integr Neurosci, 2013. 7: p. 25.

2. Li, L.F., et al., Reduced Consolation Behaviors in Physically Stressed Mandarin Voles: Involvement of Oxytocin, Dopamine D2, and Serotonin 1A Receptors Within the Anterior Cingulate Cortex. Int J Neuropsychopharmacol, 2020. 23(8): p. 511-523.

3. Carhart-Harris, R.L. and D.J. Nutt, Serotonin and brain function: a tale of two receptors. J Psychopharmacol, 2017. 31(9): p. 1091-1120.

4. Carson, W.H. and H. Kitagawa, Drug development for anxiety disorders: new roles for atypical antipsychotics. Psychopharmacol Bull, 2004. 38(1): p. 38-45.

5. Puig, M.V. and A.T. Gulledge, Serotonin and prefrontal cortex function: neurons, networks, and circuits. Mol Neurobiol, 2011. 44(3): p. 449-64.

https://doi.org/10.7554/eLife.67638.sa2

Article and author information

Author details

  1. Laifu Li

    1. Institute of Brain and Behavioral Sciences, College of Life Sciences, Shaanxi Normal University, Xi’an, China
    2. College of Life Sciences, Nanyang Normal University, Nanyang, China
    Contribution
    Investigation, Writing - original draft, Project administration
    Contributed equally with
    Li-Zi Zhang
    Competing interests
    No competing interests declared
  2. Li-Zi Zhang

    Institute of Brain and Behavioral Sciences, College of Life Sciences, Shaanxi Normal University, Xi’an, China
    Contribution
    Data curation, Methodology
    Contributed equally with
    Laifu Li
    Competing interests
    No competing interests declared
  3. Zhi-Xiong He

    Institute of Brain and Behavioral Sciences, College of Life Sciences, Shaanxi Normal University, Xi’an, China
    Contribution
    Supervision, Investigation, Methodology
    Competing interests
    No competing interests declared
  4. Huan Ma

    Institute of Brain and Behavioral Sciences, College of Life Sciences, Shaanxi Normal University, Xi’an, China
    Contribution
    Investigation, Visualization, Methodology
    Competing interests
    No competing interests declared
  5. Yu-Ting Zhang

    Institute of Brain and Behavioral Sciences, College of Life Sciences, Shaanxi Normal University, Xi’an, China
    Contribution
    Investigation, Visualization, Methodology
    Competing interests
    No competing interests declared
  6. Yu-Feng Xun

    Institute of Brain and Behavioral Sciences, College of Life Sciences, Shaanxi Normal University, Xi’an, China
    Contribution
    Validation, Investigation, Visualization
    Competing interests
    No competing interests declared
  7. Wei Yuan

    1. Institute of Brain and Behavioral Sciences, College of Life Sciences, Shaanxi Normal University, Xi’an, China
    2. Provincial Key Laboratory of Acupuncture and Medications, Shaanxi University of Chinese Medicine, Xianyang, China
    Contribution
    Validation, Investigation
    Competing interests
    No competing interests declared
  8. Wen-Juan Hou

    Institute of Brain and Behavioral Sciences, College of Life Sciences, Shaanxi Normal University, Xi’an, China
    Contribution
    Investigation, Visualization
    Competing interests
    No competing interests declared
  9. Yi-Tong Li

    Institute of Brain and Behavioral Sciences, College of Life Sciences, Shaanxi Normal University, Xi’an, China
    Contribution
    Validation, Investigation
    Competing interests
    No competing interests declared
  10. Zi-Jian Lv

    Institute of Brain and Behavioral Sciences, College of Life Sciences, Shaanxi Normal University, Xi’an, China
    Contribution
    Supervision, Visualization
    Competing interests
    No competing interests declared
  11. Rui Jia

    Institute of Brain and Behavioral Sciences, College of Life Sciences, Shaanxi Normal University, Xi’an, China
    Contribution
    Supervision, Visualization
    Competing interests
    No competing interests declared
  12. Fa-Dao Tai

    Institute of Brain and Behavioral Sciences, College of Life Sciences, Shaanxi Normal University, Xi’an, China
    Contribution
    Resources, Supervision, Writing - review and editing
    For correspondence
    taifadao@snnu.edu.cn
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6804-4179

Funding

National Natural Science Foundation of China (31970424)

  • Fa-Dao Tai

Natural Science Foundation of Shaanxi Province (2018JM3032)

  • Fa-Dao Tai

Fundamental Research Funds for the Central Universities (GK201903059)

  • Fa-Dao Tai

National Natural Science Foundation of China (31670421)

  • Fa-Dao Tai

National Natural Science Foundation of China (31372213)

  • Fa-Dao Tai

National Natural Science Foundation of China (31772473)

  • Fa-Dao Tai

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank Yu-Ying Yang, Xin Zhang, and Yi-Xin Feng for assistance in conducting experiments and caring for voles. The authors declare no conflict of interest.

Ethics

Animal experimentation: All the breeding, housing, and experimental procedures in this study were in accordance with Chinese guidelines for the care and use of laboratory animals and were approved by the Animal Care and Use Committee of Shaanxi Normal University (SNNU_20190501001). All efforts were made to minimize suffering and the number of animals used during the studies.

Senior Editor

  1. Kate M Wassum, University of California, Los Angeles, United States

Reviewing Editor

  1. Peggy Mason, University of Chicago, United States

Reviewers

  1. Peggy Mason, University of Chicago, United States
  2. JP Burkett, Emory University, United States
  3. Christian Keysers, Netherlands Institute for Neuroscience, Netherlands

Publication history

  1. Received: February 17, 2021
  2. Accepted: June 2, 2021
  3. Accepted Manuscript published: June 3, 2021 (version 1)
  4. Version of Record published: June 18, 2021 (version 2)

Copyright

© 2021, Li et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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