Separable dorsal raphe dopamine projections mimic the facets of a loneliness-like state

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This study dissects the function of 3 outputs of a specific population of modulatory neurons, dorsal raphe dopamine neurons, in social and affective behavior. It provides valuable information that both confirms prior results and provides new insights. The strength of the evidence is convincing, based on cutting-edge approaches and analysis. This study will be of interest to behavioral and systems neuroscientists, especially those interested in social and emotional behavior.

https://doi.org/10.7554/eLife.105955.3.sa0

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Abstract

Affiliative social connections facilitate well-being and survival in numerous species. Engaging in social interactions requires positive or negative motivational drive, elicited through coordinated activity across neural circuits. However, the identity, interconnectivity, and functional encoding of social information within these circuits remains poorly understood. Here, we focus on downstream projections of dorsal raphe nucleus (DRN) dopamine neurons (DRN^DAT) in mice, which we previously implicated in social motivation alongside an aversive affective state. We show that three prominent DRN^DAT projections – to the bed nucleus of the stria terminalis (BNST), central amygdala (CeA), and posterior basolateral amygdala (BLP) – play separable roles in behavior, despite substantial collateralization. Photoactivation of the DRN^DAT-CeA projection promoted social behavior and photostimulation of the DRN^DAT-BNST projection promoted exploratory behavior, while the DRN^DAT-BLP projection supported place avoidance, suggesting a negative affective state. Downstream regions showed diverse receptor expression, poising DRN^DAT neurons to act through dopamine, neuropeptide, and glutamate transmission. Furthermore, we show ex vivo that the effect of DRN^DAT photostimulation on downstream neuron excitability depended on region and baseline cell properties, resulting in excitatory responses in BNST cells and diverse responses in CeA and BLP. Finally, in vivo microendoscopic cellular-resolution recordings in the CeA with DRN^DAT photostimulation revealed a correlation between social behavior and neurons excited by social stimuli, suggesting that increased dopamine tone may recruit different CeA neurons to social ensembles. Collectively, these circuit features may facilitate a coordinated, but flexible, response in the presence of social stimuli that can be flexibly guided based on the internal social homeostatic need state of the individual.

Introduction

A close social network confers a survival advantage, both in the wild and in the laboratory (Yee et al., 2008; Koto et al., 2015; Silk et al., 2010). Indeed, our brains have evolved to adapt to many changing conditions, including when we are with others and when we are alone. Many neuromodulatory systems and neural circuits engaged in social behaviors may serve a distinct function when social stimuli are not present. In non-social contexts, dopamine transporter-expressing dorsal raphe nucleus (DRN^DAT) neurons can promote incentive memory expression (Lin et al., 2020), antinociception (Li et al., 2016; Meyer et al., 2009; Yu et al., 2021), fear response (Groessl et al., 2018), and arousal (Cho et al., 2017; Lu et al., 2006; Cho et al., 2021) – showing a clear role in many functions essential for survival. Moreover, DRN^DAT neurons undergo synaptic strengthening after social isolation and increase responsiveness to social stimuli, and stimulation of these neurons induces a prosocial state (Matthews et al., 2016). Strikingly, a functional imaging study in humans similarly revealed that 10 hours of social isolation heightened midbrain responses to social stimuli (Tomova et al., 2020). In mice, we further demonstrated that photostimulation of DRN^DAT neurons not only promoted social preference, but also induced place avoidance, suggesting an aversive internal state (Matthews et al., 2016). This led us to infer a role for these neurons in motivating social approach, driven by the desire to quell a negative state (Hull, 1943), and playing a role in social homeostasis (Lee et al., 2021; Matthews and Tye, 2019).

Taken together, this suggests a broad functional role for DRN^DAT neurons in motivating adaptive, survival-promoting behaviors under both social and non-social conditions. While the multi-functional role of dopamine neurons in the DRN seems clear, it is yet unclear how these cells exert their influence at a circuit level, and the question remains: how do DRN^DAT neurons simultaneously motivate social approach while also inducing a negative state consistent with place avoidance? What downstream targets receive this signal, and how do they respond?

There are several circuit motifs and neural encoding strategies that could enable DRN^DAT neurons to simultaneously regulate these behavioral states and motivate adaptive responses. In a drive-state sequence model, if these DRN^DAT neurons were the control center in the social homeostat (Lee et al., 2021; Matthews and Tye, 2019), the unpleasant state of being isolated could then feed forward in a sequential chain to induce motivation to rectify this social deficit. However, in an effector state activation model, many parallel actions may be taken to address the challenge, and a pervasive behavioral state may be triggered by a neuromodulatory broadcast signal. In a parallel circuit model, distinct functional roles may be associated with projection-defined subpopulations in parallel (e.g. Han et al., 2017; Kim et al., 2013; Kohl et al., 2018; Lammel et al., 2011; Namburi et al., 2015; Senn et al., 2014; Tye et al., 2011), and neurons may simultaneously encode multiple types of information (i.e. exhibit ‘mixed selectivity’ Rigotti et al., 2013; Tian et al., 2016) or behavioral output may be governed by context- or state-dependency (e.g. Krzywkowski et al., 2020; Kyriazi et al., 2018; Lemos et al., 2012; Seo et al., 2019). Yet, the mechanisms through which DRN^DAT neurons exert their influence over social behavior have yet to be unraveled.

Here, we addressed the question of how DRN^DAT neurons modulate both sociability and valence by exploring the functional role and anatomical targets of distinct DRN^DAT projections in mice. We show that parallel DRN^DAT projections to different targets play separable roles in behavior, in spite of their heavily-collateralizing anatomical arrangement. Downstream, we find that within DRN^DAT terminal fields, there is spatial segregation of dopamine and neuropeptide receptor expression. Furthermore, photostimulation of DRN^DAT inputs can modulate downstream neuronal excitability depending on their baseline cell properties. Lastly, we find that DRN^DAT input enables a shift in central amygdala dynamics that allows it to predict social preference. These findings highlight the anatomical and functional heterogeneity that exists at multiple levels within the DRN^DAT system. We suggest this organization may underlie the capacity of the DRN^DAT system to exert a broad influence over different forms of behavior: allowing coordinated control over downstream neuronal activity and across the brain to signal a behavioral state that mimics a loneliness-like phenotype.

Results

DRN^DAT neurons project to and exhibit dense collateralization to distinct subregions of the amygdala and extended amygdala

To explore the circuit motifs (Tye, 2018) and computational implementation (Lockwood et al., 2020) through which the DRN^DAT system might operate, we examined whether discrete DRN^DAT projections underlie distinct features of behavior. Prominent DRN^DAT projections were identified by quantifying downstream fluorescence following Cre-dependent expression of eYFP in DAT^IREScre (B6.SJL-^{Slc6a3tm1.1(cre)Bkmn/J}) mice (Matthews et al., 2016; Bäckman et al., 2006; Cardozo Pinto et al., 2019; Lammel et al., 2015; Figure 1—figure supplement 1). We observed a distinct pattern of innervation arising from the ventral tegmental area (VTA)^DAT and DRN^DAT subpopulations (Figure 1A–D), with DRN^DAT projections most densely targeting the oval nucleus of the BNST (ovBNST) and lateral nucleus of the central amygdala (CeL). We also observed weaker, but significant, input to the posterior part of the basolateral amygdala (BLP), consistent with previous tracing studies (Lin et al., 2020; Cardozo Pinto et al., 2019; Hasue and Shammah-Lagnado, 2002; Meloni et al., 2006; Oh et al., 2014). Given that the extended amygdala and basolateral amygdala complex have been implicated in aversion- (Davis et al., 2010; Goode and Maren, 2017; Janak and Tye, 2015; Lebow and Chen, 2016) and reward-related processes (Namburi et al., 2015; Douglass et al., 2017; Jennings et al., 2013; Kim et al., 2017; Tye et al., 2008; Tye et al., 2010; Bayless et al., 2023), and connect with hindbrain motor nuclei to elicit autonomic and behavioral changes, we focused on these DRN^DAT projections (Figure 1D).

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DRN^DAT and VTA^DAT afferents target distinct downstream regions.

(A) Example images of downstream regions showing TH expression from immunohistochemistry. (B) eYFP expression in the prefrontal cortex (PFC), nucleus accumbens (NAc), bed nucleus of the stria terminalis (BNST), central amygdala (CeA), and posterior basolateral amygdala (BLP) following injection into the DRN (upper panels) and the VTA (lower panels). (C) Quantification of mean eYFP fluorescence in subregions from each structure (PFC: n=18 and 14 sections, striatum: n=20 and 21 sections, BNST: n=14 and 13 sections, CeA: n=24 and 27 sections, amygdala: n=45 and 51 sections from DRN and VTA injections, respectively, from 6 mice). eYFP fluorescence was significantly greater following VTA injection in all striatal subregions (unpaired t-test: CPu: t₃₉=13.23, p<0.0001; NAc core: t₃₉=13.56, p<0.0001; NAc lateral shell: t₃₁=13.01, p<0.0001; NAc medial shell: t₃₇=4.49, p<0.0001), and significantly greater following DRN injection in the BNST oval nucleus (unpaired t-test: t₂₂=3.95, p=0.0007) and CeA lateral division (unpaired t-test: t₃₄=3.18, p=0.0031). (D) Images from three selected downstream targets showing average terminal density in the middle anteroposterior (AP) region following eYFP expression in DRN^DAT (left) or VTA^DAT (right) neurons. (E) The retrograde tracer cholera toxin subunit-B (CTB) conjugated to Alexa Fluor 555 (CTB-555, pseudo-colored magenta) or Alexa-Fluor 647 (CTB-647, pseudo-colored cyan) was injected into two downstream targets. (F) Confocal images showing representative injection sites for dual BNST and CeA injections (left panels), BNST and BLP (center panels), and CeA and BLP (right panels). (G) High-magnification images of DRN cells expressing CTB-555 (magenta), CTB-647 (cyan), and TH (green) following injection into the BNST and CeA. White arrows indicate triple-labeled cells. (H) Venn diagrams showing the proportion of CTB+/TH + cells in the DRN following dual injections placed in the BNST and CeA (left), BNST and BLP (center), or CeA and BLP (right). When injections were placed in the BNST and CeA, dual CTB-labeled TH + cells constituted 46% of all BNST projectors and 55% of all CeA projectors. In contrast, when injections were placed in the BNST and BLP, or CeA and BLP, the proportion of dual-labeled cells was considerably lower (7.6% of BNST projectors and 9.7% of CeA projectors). Bar graphs show mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. PFC: Cg = cingulate cortex, PL = prelimbic cortex, IL = infralimbic cortex; striatum: CPu = caudate putamen, NAc core = nucleus accumbens core, NAc l.sh.=nucleus accumbens lateral shell, NAc m.sh.=nucleus accumbens medial shell; BNST: oval nuc.=BNST oval nucleus, lat.=BNST lateral division, med.=BNST medial division, vent.=BNST ventral part; CeL = central amygdala lateral division, CeM = central amygdala medial division, CeC = central amygdala capsular division; amygdala: LA = lateral amygdala, BLA = basolateral amygdala, BLP = basolateral amygdala posterior.

Figure 1—source data 1 Mean DRN^DAT eYFP fluorescence in downstream regions, as shown in Figure 1C.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig1-data1-v1.csv
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Figure 1—source data 2 Colocalization counts of CTB+/TH + cells in the DRN, as shown in Figure 1H.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig1-data2-v1.csv
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We next considered the anatomical organization of these projections to determine whether form gives rise to function. In other words, we investigated whether DRN^DAT outputs exhibit a circuit arrangement that facilitates a coordinated behavioral response. Axonal collateralization is one circuit feature that facilitates coordinated activity across broadly distributed structures (Rockland, 2018). Although VTA^DAT projections to striatal and cortical regions typically show little evidence of collateralization (Aransay et al., 2015; Beier et al., 2015; Lerner et al., 2015; Matsuda et al., 2009; Moore and Bloom, 1978), in contrast, DRN serotonergic neurons collateralize heavily to innervate the prefrontal cortex, striatum, midbrain, and amygdala (Gagnon and Parent, 2014; van der Kooy and Hattori, 1980; Waselus et al., 2011). However, it has yet to be determined whether DRN^DAT neurons are endowed with this property.

To assess whether DRN^DAT neurons exhibit axon collaterals, we performed dual retrograde tracing with fluorophore-conjugated cholera toxin subunit B (CTB; Beyeler et al., 2018). We injected each tracer into two of the three downstream sites (BNST, CeA, and/or BLP; Figure 1E, F, Figure 1—figure supplement 2A–C) and, after 7 days for retrograde transport, we examined CTB-expressing cells in the DRN that were co-labeled with tyrosine hydroxylase (TH; Figure 1G). CTB injections into the BNST and CeA resulted in numerous TH + cells labeled with both CTB-conjugated fluorophores, but fewer dual-labeled cells were observed when injections were placed in the BNST and BLP, or CeA and BLP (Figure 1H, Figure 1—figure supplement 2D, E). These data suggest significant collateralization to the extended amygdala, which includes the BNST and CeA (Lin et al., 2020; Janak and Tye, 2015). To confirm the presence of axon collaterals, we employed an intersectional viral strategy to selectively label CeA-projecting DRN^DAT neurons with cytoplasmic eYFP (Figure 1—figure supplement 2F, G). This resulted in eYFP-expressing terminals both in the CeA and in the BNST (Figure 1—figure supplement 2H, I).

DRN^DAT-BLP photostimulation promotes place avoidance

We next considered whether DRN^DAT projections to the BNST, CeA, and BLP play separable or overlapping functional roles in modulating behavior. VTA dopaminergic input to the BNST and CeA has been implicated in threat discrimination (De Bundel et al., 2016; Jo et al., 2018), anxiety-related behavior (de la Mora et al., 2012), and drug-induced reward (Eiler et al., 2003; Epping-Jordan et al., 1998; Rezayof et al., 2002; Thiel et al., 2010), while in the BLA complex, dopamine signaling supports both fear (Bissière et al., 2003; Fadok et al., 2009; Guarraci et al., 1999; de Oliveira et al., 2011) and appetitive learning (Tye et al., 2010; Lutas et al., 2019). However, the question remains: do the same DRN^DAT projection neurons mediate different facets of a loneliness-like state, such as aversion, vigilance, and social motivation?

To test the hypothesis that distinct DRN^DAT projections promote sociability, vigilance, and place avoidance (Matthews et al., 2016), we performed projection-specific ChR2-mediated photostimulation. We injected an AAV enabling Cre-dependent expression of ChR2 into the DRN of DAT::Cre male mice and implanted optic fibers over the BNST, CeA, or BLP (Figure 2A, Figure 2—figure supplement 1A–F). Given that we previously observed that behavioral effects of DRN^DAT photostimulation were predicted by an animal’s social rank (Matthews et al., 2016), we also assessed relative social dominance using the tube test (Lindzey et al., 1961; Wang et al., 2011; Zhou et al., 2018b) prior to behavioral assays and photostimulation (Figure 2A, Figure 2—figure supplement 1G, H).

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DRN^DAT-BLP (but not DRN^DAT-BNST or DRN^DAT-CeA) photostimulation promotes place avoidance.

(A) AAV₅-DIO-ChR2-eYFP or AAV₅-DIO-eYFP was injected into the DRN of DAT::Cre mice and optic fibers implanted over the BNST, CeA, or BLP to photostimulate DRN^DAT terminals. After >7 weeks, viral expression cages of mice were assayed for social dominance using the tube test, prior to other behavioral tasks. (**B–D**) Left panels: example tracks of DRN^DAT-BNST:ChR2, DRN^DAT-CeA:ChR2, and DRN^DAT-BLP:ChR2 mice in the real-time place preference (RTPP) assay. Right panels: bar graphs showing the difference in % time spent in the stimulated (‘ON’) and unstimulated (‘OFF’) zones. There were no significant RTPP differences detected in (B) DRN^DAT-BNST:ChR2 (DRN^DAT-BNST:ChR2: N=29 mice, DRN^DAT-BNST:eYFP: N=14 mice; unpaired t-test: t₄₁=1.44, p=0.156) and (C) DRN^DAT-CeA:ChR2 mice (DRN^DAT-CeA:ChR2: N=28 mice, DRN^DAT-CeA:eYFP: N=13 mice; unpaired t-test: t₃₉=0.828, p=0.413) compared to their respective eYFP control mice groups. However, (D) DRN^DAT-BLP:ChR2 mice spent proportionally less time in the stimulated zone relative to DRN^DAT-BLP:eYFP mice (DRN^DAT-BLP:ChR2: N=14 mice, DRN^DAT-BLP:eYFP: N=8 mice; unpaired t-test: t₂₀=2.13, p=0.0455). (**E–G**) Time spent in the ON zone across the 30 min session. (G) DRN^DAT-BLP:ChR2 mice spent significantly less time in the ON zone relative to DRN^DAT-BLP:eYFP mice (DRN^DAT-BLP:ChR2: N=14 mice, DRN^DAT-BLP:eYFP: N=8 mice; repeated measures two-way ANOVA: F_1,20 = 4.53, main effect of opsin p=0.046). (**H–J**) Scatter plots showing relative dominance plotted against the difference in zone time (insets show mean values for subordinate, intermediate, and dominant mice) for (H) DRN^DAT-BNST, (I) DRN^DAT-CeA, or (J) DRN^DAT-BLP mice. Bar and line graphs display mean ± SEM. *p<0.05.

Figure 2—source data 1 DRN^DAT-BNST:ChR2 RTPP percent time difference (ON-OFF), as shown in Figure 2B.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig2-data1-v1.csv
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Figure 2—source data 2 DRN^DAT-CeA:ChR2 RTPP percent time difference (ON-OFF), as shown in Figure 2C.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig2-data2-v1.csv
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Figure 2—source data 3 DRN^DAT-BLP:ChR2 RTPP percent time difference (ON-OFF), as shown in Figure 2D.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig2-data3-v1.csv
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Figure 2—source data 4 DRN^DAT-BNST:ChR2 RTPP percent time in ON (binned), as shown in Figure 2E.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig2-data4-v1.csv
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Figure 2—source data 5 DRN^DAT-CeA:ChR2 RTPP percent time in ON (binned), as shown in Figure 2F.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig2-data5-v1.csv
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Figure 2—source data 6 DRN^DAT-BLP:ChR2 RTPP percent time in ON (binned), as shown in Figure 2G.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig2-data6-v1.csv
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Figure 2—source data 7 DRN^DAT-BNST:ChR2 RTPP percent time difference (ON-OFF) x relative dominance, as shown in Figure 2H.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig2-data7-v1.csv
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Figure 2—source data 8 DRN^DAT-CeA:ChR2 RTPP percent time difference (ON-OFF) x relative dominance, as shown in Figure 2I.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig2-data8-v1.csv
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Figure 2—source data 9 DRN^DAT-BLP:ChR2 RTPP percent time difference (ON-OFF) x relative dominance, as shown in Figure 2J.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig2-data9-v1.csv
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We first assessed whether photostimulation was sufficient to support place preference using the real-time place-preference (RTPP) assay. Here, we found that photostimulation of the DRN^DAT-BLP projection, but not the projection to the BNST or CeA, produced avoidance of the stimulation zone, relative to eYFP controls (Figure 2B–G). However, we did not find a significant correlation between social dominance and the magnitude of this effect (Figure 2H–J). Importantly, we did not detect an effect of photostimulation of DRN^DAT projections on operant intracranial self-stimulation (Figure 2—figure supplement 2).

DRN^DAT-BNST photostimulation promotes non-social exploration

Next, we considered whether DRN^DAT projections to the BNST, CeA, or BLP play a role in increasing vigilance, a common behavioral marker in individuals experiencing loneliness (Cacioppo et al., 2016; Cacioppo and Hawkley, 2009). To assess how projection-specific photostimulation of DRN^DAT terminals affected exploratory behavior, we used the open field test (OFT) and elevated plus maze (EPM). While we found no effect of optical stimulation of DRN^DAT terminals on locomotion or time in center in the OFT (Figure 3—figure supplement 1), we found stimulation of DRN^DAT terminals in the BNST (but not in the CeA or BLP) resulted in a weak trend toward increased time spent in the open arm of the EPM (Figure 3A–C), which can be interpreted as exploratory behavior linked with a vigilant state (Rodgers and Dalvi, 1997). However, we found no correlation between social dominance and open arm time (Figure 3D–F). Strikingly, during social interaction with a novel juvenile in the home cage, we found that photoactivation of the DRN^DAT-BNST projection increased rearing behavior (a form of nonsocial exploration Bailey and Crawley, 2009; Lever et al., 2006; Figure 3G–L), an effect that was not previously observed with cell body photostimulation (Matthews et al., 2016). However, we did not find a significant correlation between social dominance and the expression of optically induced rearing behavior (Figure 3J–L).

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DRN^DAT-BNST (but not DRN^DAT-CeA or DRN^DAT-BLP) photostimulation promotes non-social exploratory behavior.

(**A–C**) Left panels: example tracks in the elevated plus maze (EPM) from a (A) DRN^DAT-BNST:ChR2, (B), DRN^DAT-CeA:ChR2, and (C), DRN^DAT-BLP:ChR2 mouse. Upper right panels: time spent in the open arms of the EPM across the 15 min session. Photostimulation had no significant effect on time spent in the open arms of the EPM (two-way ANOVA, light x group interaction, BNST – F_2,50=2.008, p=0.145, CeA – F_2,72=0.118, p=0.889, BLP – F_2,40=0.354, p=0.704) for (A) DRN^DAT-BNST, (B), DRN^DAT-CeA, or (C) DRN^DAT-BLP mice. Bottom right panels: difference in time spent in open arms of the EPM between the stimulation ON and first OFF epochs. Photostimulation had no significant effect on time spent in the open arms of the EPM for (A) DRN^DAT-BNST (DRN^DAT-BNST:ChR2: N=19 mice, DRN^DAT-BNST:eYFP: N=10 mice; unpaired t-test: t₂₇=1.39, p=0.177), (B) DRN^DAT-CeA (DRN^DAT-CeA:ChR2: N=23 mice, DRN^DAT-CeA:eYFP: N=14 mice; unpaired t-test: t₃₅=0.639, p=0.527), or (C) DRN^DAT-BLP mice (DRN^DAT-BLP:ChR2: N=14 mice, DRN^DAT-BLP:eYFP: N=8 mice; unpaired t-test: t₂₀=0.759, p=0.457). (**D–F**) Scatter plots showing relative dominance plotted against the difference in the open arm zone time (insets show mean values for subordinate, intermediate, and dominant mice) for (D) DRN^DAT-BNST, (E) DRN^DAT-CeA, or (F) DRN^DAT-BLP mice. (**G–I**) Home-cage behavior was assessed in the juvenile intruder assay across two counterbalanced sessions, one paired with photostimulation (‘ON’) and one without (‘OFF’) for (G) DRN^DAT-BNST, (H) DRN^DAT-CeA, or (I) DRN^DAT-BLP mice. DRN^DAT-BNST photostimulation increased time spent rearing (DRN^DAT-BNST:ChR2: N=24 mice, DRN^DAT-BNST:eYFP: N=13 mice; paired t-test: t₂₃=2.32, p=0.0298), but DRN^DAT-CeA and DRN^DAT-BLP photostimulation did not. (**J–L**) Scatter plots showing relative dominance plotted against the difference in rearing time with optical stimulation (ON-OFF) (insets show mean values for subordinate, intermediate, and dominant mice) for (J) DRN^DAT-BNST, (K) DRN^DAT-CeA, or (L) DRN^DAT-BLP mice. Bar and line graphs display mean ± SEM. *p<0.05.

Figure 3—source data 1 DRN^DAT-BNST:ChR2 EPM open arm time (binned), as shown in Figure 3A.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig3-data1-v1.csv
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Figure 3—source data 2 DRN^DAT-CeA:ChR2 EPM open arm time (binned), as shown in Figure 3B.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig3-data2-v1.csv
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Figure 3—source data 3 DRN^DAT-BLP:ChR2 EPM open arm time (binned), as shown in Figure 3C.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig3-data3-v1.csv
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Figure 3—source data 4 DRN^DAT-BNST:ChR2 EPM open arm time (ON-OFF) x relative dominance, as shown in Figure 3D.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig3-data4-v1.csv
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Figure 3—source data 5 DRN^DAT-CeA:ChR2 EPM open arm time (ON-OFF) x relative dominance, as shown in Figure 3E.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig3-data5-v1.csv
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Figure 3—source data 6 DRN^DAT-BLP:ChR2 EPM open arm time (ON-OFF) x relative dominance, as shown in Figure 3F.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig3-data6-v1.csv
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Figure 3—source data 7 DRN^DAT-BNST:ChR2 juvenile intruder time spent rearing, as shown in Figure 3G.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig3-data7-v1.csv
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Figure 3—source data 8 DRN^DAT-CeA:ChR2 juvenile intruder time spent rearing, as shown in Figure 3H.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig3-data8-v1.csv
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Figure 3—source data 9 DRN^DAT-BLP:ChR2 juvenile intruder time spent rearing, as shown in Figure 3I.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig3-data9-v1.csv
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Figure 3—source data 10 DRN^DAT-BNST:ChR2 juvenile intruder time spent rearing (ON-OFF) x relative dominance, as shown in Figure 3J.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig3-data10-v1.csv
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Figure 3—source data 11 DRN^DAT-CeA:ChR2 juvenile intruder time spent rearing (ON-OFF) x relative dominance, as shown in Figure 3K.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig3-data11-v1.csv
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Figure 3—source data 12 DRN^DAT-BLP:ChR2 juvenile intruder time spent rearing (ON-OFF) x relative dominance, as shown in Figure 3L.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig3-data12-v1.csv
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DRN^DAT-CeA photostimulation promotes sociability

To assess how projection-specific photostimulation of DRN^DAT terminals affected social preference, we used the three-chamber sociability task (Moy et al., 2004), where group-housed mice freely explored a chamber containing a novel juvenile mouse and a novel object at opposite ends (Figure 4A–C). This revealed that optical stimulation of the DRN^DAT-CeA projection increased social preference, but no significant effect was observed with photostimulation of either the DRN^DAT-BNST or DRN^DAT-BLP projections (Figure 4D–F; Figure 4—figure supplement 1). Furthermore, we found that the optically induced change in social preference in DRN^DAT-CeA mice was positively correlated with social dominance, suggesting that photostimulation elicited a greater increase in sociability in dominant mice (Figure 4G–I). This emulates the previous association found with photostimulation at the cell body level and social dominance (Matthews et al., 2016).

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DRN^DAT-CeA (but not DRN^DAT-BNST or DRN^DAT-BLP) photostimulation promotes sociability in a rank-dependent manner.

(**A–C**) Heatmaps showing the relative location of ChR2-expressing mice in the three-chamber sociability assay, with optic fibers located over the (A) BNST, (B) CeA, or (C) BLP. The task was repeated across 2 days, with one session paired with photostimulation (‘ON’) and one without (‘OFF’). (**D–F**) Bar graphs showing social preference in three-chamber sociability assay. (D) Photostimulation of DRN^DAT-BNST terminals (8 pulses of 5ms pulse-width 473 nm light, delivered at 30 Hz every 5 s) in ChR2-expressing mice (DRN^DAT-BNST:ChR2) had no significant effect on time spent in the social zone relative to the object zone (DRN^DAT-BNST:ChR2: N=27 mice, DRN^DAT-BNST:eYFP: N=14 mice; ‘social:object ratio’; paired t-test: t₂₆=0.552, p=0.586), (E) but increased social:object ratio for DRN^DAT-CeA:ChR2 mice (DRN^DAT-CeA:ChR2: N=29 mice, DRN^DAT-CeA:eYFP: N=13 mice; paired t-test: t₂₈=2.91; corrected for multiple comparisons: p=0.021) (F) and had no significant effect for DRN^DAT-BLP:ChR2 mice (DRN^DAT-BLP:ChR2: N=14 mice, DRN^DAT-BLP:eYFP: N=7 mice; paired t-test: t₁₃=1.62, p=0.130). (**G–I**) Scatter plots displaying relative dominance plotted against the change in social zone time with optical stimulation (ON-OFF) for (G) DRN^DAT-BNST, (H) DRN^DAT-CeA, or (I) DRN^DAT-BLP mice, showing significant positive correlation in DRN^DAT-CeA:ChR2 mice (Pearson’s correlation: r=0.549, p=0.002, N=29 mice). Inset bar graphs show mean values for subordinate, intermediate, and dominant mice. (**J–L**) Home-cage behavior was assessed in the juvenile intruder assay across two counterbalanced sessions, one paired with photostimulation (‘ON’) and one without (‘OFF’) for (J) DRN^DAT-BNST, (K) DRN^DAT-CeA, or (L) DRN^DAT-BLP mice. DRN^DAT-CeA photostimulation in ChR2-expressing mice increased time spent engaged in face investigation with the juvenile mouse (DRN^DAT-CeA:ChR2: N=22 mice, DRN^DAT-CeA:eYFP: N=14 mice; paired t-test: t₂₂=2.36, p=0.027). (**M–O**) Scatter plots showing relative dominance plotted against the difference in face investigation time with optical stimulation (ON-OFF) (insets show mean values for subordinate, intermediate, and dominant mice) for (M) DRN^DAT-BNST, (N) DRN^DAT-CeA, or (O) DRN^DAT-BLP mice. (P) A two-state Markov model was used to examine behavioral transitions during the juvenile intruder assay for DRN^DAT-CeA mice. (**Q, R**) Bar graphs showing the difference in transition probability (ON-OFF) for (Q) within-state transitions and (R) across-state transitions, for DRN^DAT-CeA:ChR2 and DRN^DAT-CeA:eYFP mice. There was no significant difference between ChR2 and eYFP groups for the change in within-state transition probability (DRN^DAT-CeA:ChR2: N=22 mice, DRN^DAT-CeA:eYFP: N=14 mice; two-way ANOVA: opsin x transition interaction, F_1,68=3.385, p=0.0702), (R) but there was a significant interaction between opsin and across-state transition probability (DRN^DAT-CeA:ChR2: N=22 mice, DRN^DAT-CeA:eYFP: N=14 mice; two-way ANOVA: opsin x transition interaction, F_1,68=4.452, p=0.0385) with photostimulation. Bar and line graphs display mean ± SEM. *p<0.05, **p<0.01.

Figure 4—source data 1 DRN^DAT-BNST:ChR2 three-chamber social:object ratio, as shown in Figure 4D.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig4-data1-v1.csv
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Figure 4—source data 2 DRN^DAT-CeA:ChR2 three-chamber social:object ratio, as shown in Figure 4E.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig4-data2-v1.csv
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Figure 4—source data 3 DRN^DAT-BLP:ChR2 three-chamber social:object ratio, as shown in Figure 4F.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig4-data3-v1.csv
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Figure 4—source data 4 DRN^DAT-BNST:ChR2 time spent in social zone (ON-OFF) x relative dominance, as shown in Figure 4G.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig4-data4-v1.csv
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Figure 4—source data 5 DRN^DAT-CeA:ChR2 time spent in social zone (ON-OFF) x relative dominance, as shown in Figure 4H.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig4-data5-v1.csv
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Figure 4—source data 6 DRN^DAT-BLP:ChR2 time spent in social zone (ON-OFF) x relative dominance, as shown in Figure 4I.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig4-data6-v1.csv
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Figure 4—source data 7 DRN^DAT-BNST:ChR2 juvenile intruder time spent in face investigation, as shown in Figure 4J.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig4-data7-v1.csv
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Figure 4—source data 8 DRN^DAT-CeA:ChR2 juvenile intruder time spent in face investigation, as shown in Figure 4K.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig4-data8-v1.csv
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Figure 4—source data 9 DRN^DAT-BLP:ChR2 juvenile intruder time spent in face investigation, as shown in Figure 4L.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig4-data9-v1.csv
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Figure 4—source data 10 DRN^DAT-BNST:ChR2 juvenile intruder time spent in face investigation (ON-OFF) x relative dominance, as shown in Figure 4M.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig4-data10-v1.csv
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Figure 4—source data 11 DRN^DAT-CeA:ChR2 juvenile intruder time spent in face investigation (ON-OFF) x relative dominance, as shown in Figure 4N.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig4-data11-v1.csv
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Figure 4—source data 12 DRN^DAT-BLP:ChR2 juvenile intruder time spent in face investigation (ON-OFF) x relative dominance, as shown in Figure 4O.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig4-data12-v1.csv
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Figure 4—source data 13 DRN^DAT-CeA:ChR2 juvenile intruder markov model (transition within state), as shown in Figure 4Q.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig4-data13-v1.csv
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Figure 4—source data 14 DRN^DAT-CeA:ChR2 juvenile intruder markov model (transition across states), as shown in Figure 4R.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig4-data14-v1.csv
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Next, to gain further insight into the functional divergence of DRN^DAT projections in ethological behaviors, we assessed the effects of photostimulation on social interaction with a novel juvenile in the home cage. Here, photoactivation of the DRN^DAT-CeA projection modestly increased face sniffing of the juvenile mouse, consistent with a pro-social role for this projection (Figure 4J–L), although no correlation between optically induced change in face sniffing and social dominance was observed (Figure 4M–O). When we plotted the difference score (ON-OFF) for face sniffing against rearing (ON-OFF; Figure 4—figure supplement 2A–C), we observed that DRN^DAT-BNST mice tended to engage in more rearing and less face sniffing during photostimulation (i.e. located in the upper left quadrant), whereas DRN^DAT-CeA mice tended to exhibit less rearing and more face sniffing during photostimulation (i.e. located in the lower right quadrant).

To explore the relationship between social dominance and baseline behavioral profile, we applied a data-driven approach by examining behavioral measures obtained from different assays in a correlation matrix (Figure 4—figure supplement 2D). This showed a weak, negative correlation between social dominance and open arm time in the elevated plus maze (EPM) – consistent with a previous report of higher trait anxiety in dominant mice (Larrieu et al., 2017). However, social dominance did not correlate significantly with any other behavioral variable. Additionally, our analysis of baseline behavioral profile revealed a robust negative correlation between the time spent engaged in social sniffing and time spent rearing (Figure 4—figure supplement 2D). Furthermore, following dimensionality reduction on baseline behavioral variables, we did not find clearly differentiated clusters of high- and low-ranked mice (Figure 4—figure supplement 2E), suggesting that the variation governing these latent behavioral features is not related to social rank.

Finally, to determine whether DRN^DAT-CeA photostimulation affected the probability of behavioral state transition (Füzesi et al., 2016; Lee et al., 2019), we examined the sequential structure of behavior using a First-order Markov model (Lee et al., 2019; Tejada et al., 2010). Considering a two-state model consisting of ‘social’ and ‘nonsocial’ behaviors (Figure 4P), we found that photostimulation in DRN^DAT-CeA mice did not significantly change the probability of transitioning within social and nonsocial state (Figure 4Q), but did significantly change the probability of transitioning between social and nonsocial states (Figure 4R). This suggests that the DRN^DAT-CeA projection may increase engagement in social behavior by altering the overall structure of behavioral transitions.

DRN^DAT terminal fields contain spatially segregated dopamine and neuropeptide receptor populations

Our data suggest that DRN^DAT projections exert divergent effects over behavior, despite substantial overlap in their upstream cells of origin. Given this overlap, we reasoned that one mechanism through which these projections might achieve distinct behavioral effects is via differential recruitment of downstream signaling pathways. We, therefore, next considered whether the pattern of receptor expression differed within the DRN^DAT terminal field of these downstream regions.

Subsets of DRN^DAT neurons co-express vasoactive intestinal peptide (VIP) and neuropeptide-W (NPW; Dougalis et al., 2012; Huang et al., 2019; Motoike et al., 2016), and so we examined both dopamine (Drd1 and Drd2) and neuropeptide (Vipr2 and Npbwr1) receptor expression within DRN^DAT terminal fields. To achieve this, we performed single molecule fluorescence in situ hybridization (smFISH) using RNAscope (Figure 5—figure supplement 1A, B). In the BNST and CeA, we observed a strikingly similar pattern of receptor expression with dense neuropeptide receptor expression in the oval BNST and ventromedial CeL, and a high degree of co-localization (Figure 5A–H, Figure 5—figure supplement 1C–H). In the BNST and CeA subregions containing the highest density of DRN^DAT terminals, dopamine receptor expression was relatively more sparse, with Drd2 more abundant than Drd1, as previously described (Kim et al., 2017; De Bundel et al., 2016; de la Mora et al., 2012; McCullough et al., 2018a; McCullough et al., 2018b; Figure 5A–H). The DRN^DAT terminal field of the BLP displayed a markedly different receptor expression pattern, dominated by Drd1 (Figure 5I–L, Figure 5—figure supplement 1I–K), consistent with previous reports (de la Mora et al., 2012; Lutas et al., 2019; McCullough et al., 2018a). Thus, in contrast to the BNST and CeA, the effects of DRN^DAT input to the BLP may be predominantly mediated via D₁-receptor signaling. Collectively, this expression pattern suggests that the dopamine- and neuropeptide-mediated effects of DRN^DAT input may be spatially segregated within downstream regions, providing the infrastructure for divergent modulation of cellular subsets.

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Spatial segregation of dopamine and neuropeptide receptor populations within DRN^DAT terminal fields.

(A) Mean projection of terminal density in the middle anteroposterior (AP) region of the BNST, following eYFP expression in DRN^DAT (left) or VTA^DAT (right) neurons. (B) Mean projection showing fluorescent puncta in the BNST indicating detection of *Drd1* (red), *Drd2* (yellow), *Vipr2* (green), or *Npbwr1* (blue) mRNA transcripts. (C) Line graphs showing the percent of cells expressing each receptor (≥5 puncta) across AP locations for the oval nucleus, dorsolateral BNST, and dorsomedial BNST (two-way ANOVA, oval nucleus: probe x AP interaction, F_9,160=6.194, p<0.0001, dorsolateral BNST: probe x AP interaction, F_12,167=3.410, p=0.0002, dorsomedial BNST: probe x AP interaction, F_12,161=2.268, p=0.0110). *Drd1: n=*51,55,53 *Drd2: n=*52,55,53 *Vipr2: n=*37,39,37 *Npbwr1: n=*36,38,38 sections, for oval nucleus, dorsolateral BNST, and dorsomedial BNST, respectively, from 4 mice. (D) Matrices indicating overlap between mRNA-expressing cells: square shade indicates the percent of cells expressing the gene in the column from within cells expressing the gene in the row. (E) Mean projection of terminal density in the middle AP region of the CeA, following eYFP expression in DRN^DAT (left) or VTA^DAT (right) neurons. (F) Mean projection showing fluorescent puncta in the CeA indicating mRNA expression.(G) Line graphs showing the % of cells expressing each receptor (≥5 puncta) across AP locations for the CeL, CeM, and CeC (two-way ANOVA, CeL: probe x AP interaction, F_12,220=8.664, p<0.0001, CeM: main effect of probe, F_3,186=60.30, p<0.0001, CeC: probe x AP interaction, F_12,218=4.883, p<0.0001). *Drd1: n=*47,40,47 *Drd2: n=*70,55,70 *Vipr2: n=*65,57,63 *Npbwr1: n=*62,50,60 sections, for CeL, CeM, and CeC, respectively, from 4 mice. (H) Matrices indicating overlap between mRNA-expressing cells. (I) Mean projection of terminal density in the middle AP region of the BLP, following eYFP expression in DRN^DAT (left) or VTA^DAT (right) neurons. (J) Mean projection showing fluorescent puncta in the BLP indicating mRNA expression. (K) Line graphs showing the percent of cells expressing each receptor (≥5 puncta) across AP locations for the BLP and BMP (two-way ANOVA, BLP: probe x AP interaction, F_15,176=2.165, p=0.0091, BMP: main effect of probe, F_3,141=56.92, p<0.0001). *Drd1: n=*55,44 *Drd2: n=*59,46 *Vipr2: n=*41,33 *Npbwr1: n=*45,34 sections, for BLP and BMP, respectively, from 4 mice. (L) Matrices indicating overlap between mRNA-expressing cells. Line graphs show mean ± SEM.

Figure 5—source data 1 BNST RNAScope sub-regional probe expression (percent), as shown in Figure 5C.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig5-data1-v1.csv
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Figure 5—source data 2 BNST RNAScope sub-regional probe co-expression, as shown in Figure 5D.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig5-data2-v1.csv
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Figure 5—source data 3 CeA RNAScope sub-regional probe expression (percent), as shown in Figure 5G.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig5-data3-v1.csv
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Figure 5—source data 4 CeA RNAScope sub-regional probe co-expression, as shown in Figure 5H.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig5-data4-v1.csv
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Figure 5—source data 5 BLA RNAScope sub-regional probe expression (percent), as shown in Figure 5K.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig5-data5-v1.csv
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Figure 5—source data 6 BLA RNAScope sub-regional probe co-expression, as shown in Figure 5L.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig5-data6-v1.csv
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DRN^DAT input has divergent effects on downstream cellular excitability

Our data suggest that DRN^DAT projections exert divergent effects over behavior, despite substantial overlap in their upstream cells of origin. One mechanism through which these projections might achieve distinct behavioral effects is via differential modulation of activity in downstream neurons. The multi-transmitter phenotype of DRN^DAT neurons (Dougalis et al., 2012; Huang et al., 2019; Dougalis et al., 2017; Poulin et al., 2018), regionally distinct downstream receptor expression, and the observed pre- and post-synaptic actions of exogenously applied dopamine (Kash et al., 2008; Krawczyk et al., 2011; Kröner et al., 2005; Marowsky et al., 2005; Naylor et al., 2010; Rosenkranz and Grace, 1999; Rosenkranz and Grace, 2002; Silberman and Winder, 2013) provide optimal conditions for diverse modulation of neural activity. However, it remains unknown how temporally precise activation of DRN^DAT terminals influences excitability at the single-cell level.

We, therefore, next examined how DRN^DAT input affects downstream excitability. To achieve this, we expressed ChR2 in DRN^DAT neurons and used ex vivo electrophysiology to record from downstream neurons (Figure 6A–C, Figure 6—figure supplement 1A–C). Optical stimulation at the resting membrane potential evoked both excitatory and inhibitory post-synaptic potentials (EPSPs and IPSPs) in downstream cells (Figure 6D–F), which were typically monosynaptic (Figure 6—figure supplement 1D, E). During spontaneous firing, BNST cells were universally excited, whereas more diverse responses were observed with the BLP and CeA (Figure 6G–K, Figure 6—figure supplement 1F–G). The fast rise and decay kinetics of the EPSP suggest an AMPAR-mediated potential, resulting from glutamate co-release (Li et al., 2016; Matthews et al., 2016), whereas the slow IPSP kinetics are consistent with opening of GIRK channels, which can occur via D₂-receptor (Beckstead et al., 2004; Marcott et al., 2018) or GABA-_B receptor signaling (Bettler et al., 2004; Destexhe and Sejnowski, 1995; Mackay et al., 2019).

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DRN^DAT input distinctly influences downstream activity in each downstream target.

(**A–C**) In mice expressing ChR2 in DRN^DAT neurons, ex vivo electrophysiological recordings were made from (A) the BNST, (B) CeA, and (C) BLP. (**D–F**) Photostimulation of DRN^DAT terminals with blue light (8 pulses delivered at 30 Hz) evoked both excitatory and inhibitory responses at resting membrane potentials in (D) the BNST, (E) CeA, and (F) BLP. Traces show single sweeps and pie charts indicate proportion of cells with no response (‘none’), an EPSP only (‘excitation’), an IPSP only (‘inhibition’), or a mixed combination of EPSPs and IPSPs (‘mix’). Recorded cells: BNST n=19, CeA n=36, BLP n=48. (**G–I**) When constant current was injected to elicit spontaneous firing, (G) BNST cells responded to photostimulation with an increase in firing (‘excitation’), while (H) CeA and (I) BLP cells responded with an increase or a decrease in firing (‘inhibition’). Recorded cells: BNST n=5, CeA n=20, BLP n=17. (J) Properties of the optically evoked excitatory post-synaptic potential (EPSP) at resting membrane potentials – left: peak amplitude (Kruskal-Wallis statistic = 6.790, p=0.0335; Dunn’s posts-hoc tests: CeA vs BLP p=0.0378); middle: change in amplitude across light pulses; right: violin plots showing distribution of onset latencies (white circle indicates median).(K) Properties of the optically evoked inhibitory post-synaptic potential (IPSP) at resting membrane potentials – left panel: trough amplitude (one-way ANOVA, F_2,31=8.150, p=0.0014, CeA vs BLP: **p=0.0014); middle panel: violin plot showing latency to trough peak; right panel: violin plot showing tau for the current decay (white circle indicates median). (L) Workflow for agglomerative hierarchical clustering of CeA neurons and (M) BLP neurons. Four baseline electrical properties were used as input features (following max-min normalization) and Ward’s method was used to generate a cluster dendrogram, grouping cells based on Euclidean distance. (N) Dendrogram for CeA cells indicating two major clusters, with their response to DRN^DAT input indicated below each branch (excitation = black; inhibition = grey; no response = open). (O) Upper panels: cluster 1 showed baseline properties typical of ‘late-firing’ neurons and cluster 2 showed baseline properties typical of ‘regular-firing’ neurons. Lower panels: pie charts showing the response of cells in each cluster to DRN^DAT input. (P) Dendrogram for BLP cells indicating two major clusters, with their response to DRN^DAT input indicated below each branch (excitation = black; inhibition = grey; no response = open). (Q) Upper panels: cluster 1 showed baseline properties typical of pyramidal neurons and cluster 2 showed baseline properties typical of GABA interneurons. Lower panels: pie charts showing the response of cells in each cluster to DRN^DAT input. Bar and line graphs show mean ± SEM. *p<0.05, **p<0.01.

Figure 6—source data 1 BNST (resting) ex vivo responses to DRN^DAT optical stimulation, as shown in Figure 6D.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig6-data1-v1.csv
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Figure 6—source data 2 CeA (resting) ex vivo responses to DRN^DAT optical stimulation, as shown in Figure 6E.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig6-data2-v1.csv
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Figure 6—source data 3 BLP (resting) ex vivo responses to DRN^DAT optical stimulation, as shown in Figure 6F.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig6-data3-v1.csv
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Figure 6—source data 4 BNST (firing) ex vivo responses to DRN^DAT optical stimulation, as shown in Figure 6G.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig6-data4-v1.csv
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Figure 6—source data 5 CeA (firing) ex vivo responses to DRN^DAT optical stimulation, as shown in Figure 6H.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig6-data5-v1.csv
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Figure 6—source data 6 BLP (firing) ex vivo responses to DRN^DAT optical stimulation, as shown in Figure 6I.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig6-data6-v1.csv
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Figure 6—source data 7 BNST/CeA/BLP ex vivo EPSP peak amplitude in response to DRN^DAT optical stimulation, as shown in Figure 6J.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig6-data7-v1.csv
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Figure 6—source data 8 BNST/CeA/BLP ex vivo EPSP normalized amplitude in response to DRN^DAT optical stimulation, as shown in Figure 6J.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig6-data8-v1.csv
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Figure 6—source data 9 BNST/CeA/BLP ex vivo EPSP onset latency in response to DRN^DAT optical stimulation, as shown in Figure 6J.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig6-data9-v1.csv
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Figure 6—source data 10 BNST/CeA/BLP ex vivo IPSP trough amplitude in response to DRN^DAT optical stimulation, as shown in Figure 6K.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig6-data10-v1.csv
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Figure 6—source data 11 BNST/CeA/BLP ex vivo IPSP trough latency in response to DRN^DAT optical stimulation, as shown in Figure 6K.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig6-data11-v1.csv
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Figure 6—source data 12 BNST/CeA/BLP ex vivo IPSP decay tau in response to DRN^DAT optical stimulation, as shown in Figure 6K.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig6-data12-v1.csv
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Figure 6—source data 13 CeA ex vivo baseline cell properties used for hierarchical clustering, as shown in Figure 6L–O.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig6-data13-v1.csv
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Figure 6—source data 14 BLP ex vivo baseline cell properties used for hierarchical clustering, as shown in Figure 6M–Q.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig6-data14-v1.csv
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Given the diversity of responses observed in the CeA and BLP, we next examined these downstream cells in more detail. To assess whether baseline electrophysiological properties predicted the optically evoked response, we used unsupervised agglomerative hierarchical clustering to classify downstream cells (Figure 6L, M). This established approach has been successfully applied to electrophysiological datasets to reveal distinct neuronal subclasses (Cauli et al., 2000; Guthman et al., 2020; Hou et al., 2016). The resulting dendrograms yielded two major clusters in the CeA and BLP, with distinct electrophysiological characteristics (Figure 6N–Q, Figure 6—figure supplement 1H–K). CeA cells in cluster 1 represented ‘late-firing’ neurons, whereas cluster 2 was typical of ‘regular-firing’ neurons (Chieng et al., 2006; Dumont et al., 2002; Lopez de Armentia and Sah, 2004). Strikingly, these clusters exhibited dramatically different responses to DRN^DAT photostimulation, with cluster 1 ‘late-firing’ neurons excited and cluster 2 ‘regular-firing’ neurons mostly inhibited (Figure 6O). Similarly, BLP cells delineated into two major clusters, with properties characteristic of pyramidal neurons (cluster 1) and GABAergic interneurons (cluster 2; Figure 6P, Q). These clusters showed remarkably different responses to DRN^DAT input, with 93% of putative pyramidal neurons showing an inhibitory response, and 62% of putative GABAergic interneurons showing an excitatory response (Figure 6Q). In addition, clustering CeA and BLP cells together yielded a very similar result (Figure 6—figure supplement 1L–N). Thus, while photoactivation of DRN^DAT terminals elicits heterogeneous responses in downstream neurons, baseline cell properties strongly predict their response, suggesting robust synaptic organization. The opposing nature of these responses, in different neuronal subsets, suggests that – rather than inducing an overall augmentation or suppression of activity – DRN^DAT input may adjust the pattern of downstream activity, in order to exert a functional shift in behavior.

DRN^DAT input enables a functional shift in CeA dynamics to predict social preference

Our data thus far suggest that photostimulation of DRN^DAT projections to downstream extended amygdala targets elicits divergent behaviors that are, together, congruent with a loneliness-like state, with the DRN^DAT-CeA projection promoting sociability. Considering the diversity of responses in the CeA elicited by DRN^DAT input ex vivo, we next wondered how DRN^DAT input into the CeA in vivo during a behaviorally relevant task may modify how the CeA represents social information. Neuromodulatory input has been previously shown to alter responses to salient stimuli—for instance, stimulation of VTA dopamine terminals increases the signal-to-noise ratio to aversive stimuli in projection-specific populations of prefrontal cortex neurons (Vander Weele et al., 2018). However, how DRN^DAT input modifies the coding scheme of CeA neurons for social information remains unknown.

Therefore, to test the hypothesis that DRN^DAT input alters the responses of CeA neurons to functionally relevant stimuli, we examined the dynamics of CeA neurons while simultaneously stimulating DRN^DAT terminals during a three-chamber sociability task. To achieve this, we expressed the calcium indicator GCaMP7f nonspecifically in the CeA and either the red-shifted opsin ChrimsonR or a control fluorophore (TdTomato) in the DRN of DAT::Cre mice, and additionally implanted a gradient index (GRIN) lens over the CeA (Figure 7A, Figure 7—figure supplement 1A). This allowed us to stimulate DRN^DAT terminals in the CeA while resolving single-cell calcium dynamics in the CeA in vivo (Figure 7B). We confirmed ex vivo that blue light delivery alone onto DRN^DAT terminals did not elicit a ChrimsonR-mediated postsynaptic potential in CeA neurons (Figure 7—figure supplement 1B–F), and that red light delivery was still capable of eliciting ChrimsonR-mediated EPSPs and IPSPs during continuous delivery of blue light (Figure 7—figure supplement 1G–I).

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Simultaneous calcium imaging of CeA neurons and optogenetic stimulation of DRN^DAT terminals in CeA.

(A) AAV₁-hSyn-GCaMP7f was injected into the CeA and AAV₈-hSyn-FLEX-ChrimsonR-TdTomato or AAV₁-CAG-FLEX-TdTomato was injected into the DRN of DAT-Cre mice, and a GRIN lens was implanted over CeA. Experiments were conducted 7 weeks following surgery to allow adequate virus expression in axon terminals. (B) Example spatial correlation image and extracted ROIs of CeA neurons following calcium imaging processing. (C) Three chamber sociability paradigm. While group-housed, mice explored a three-chamber apparatus with a novel male juvenile stimulus on one side and a novel object stimulus on the other. During one day of the imaging experiment, DRN^DAT terminals were not stimulated, and in another session, DRN^DAT terminals were stimulated with red light delivery. Mice underwent a third imaging session, without photostimulation, following 24 hr of social isolation. (D) Mice first explored the three-chamber apparatus without social or object stimuli for a 5-min habituation period, then with the social and object stimuli for a 10-min test period. (E) Social:object ratio (left) and total social cup interaction time (right) during GH stimulation and no stimulation sessions and 24 hr isolated session in mice expressing ChrimsonR in DRN^DAT neurons. Bar and line graphs represent mean ± SEM (N=12 mice; mixed-effects model: F_1.897,30.36=0.5767, p=0.5591). (F) Representative traces from CeA calcium imaging during one three-chamber imaging session. (G) Scatter and distribution plots indicating the response strength (auROC) of recorded CeA neurons to social and object cups (GH off: n=429 cells, N=15 mice; GH on: n=441 cells, N=15 mice; SI off: n=484 cells, N=15 mice).(H) Difference in response strength (Δ auROC) of CeA neurons to social and object cups (GH off: n=429 cells; GH on: n=441 cells; SI off: n=484 cells) Kruskal-Wallis test: K-W statistic: 6.172, *p=0.0457; Dunn’s multiple comparisons test: GH off vs GH on—p=0.0580, GH off vs SI off—p>0.9999. (I) Venn diagrams showing overlap of social-encoding neurons (displaying an excitatory response, left, or an inhibitory response, right as defined with auROC) in GH off and GH on sessions (GH off and GH on co-registered neurons: n=202 cells). 16 co-registered GH off cells and 18 GH on cells exhibited an excitatory response to social stimulus with 2 cells having the same response across conditions, whereas 12 co-registered GH off and 11 GH on cells exhibited an inhibitory response with 2 cells having the same response across conditions. (J) Proportion of CeA neurons responsive to social and object cups, further classified as an excitatory (green) or inhibitory (red) response to the stimulus as defined with auROC. (K) Proportion of recorded neurons that have an excitatory or inhibitory response to the social cup and (L) to the object cup (N=12 mice). (M) Correlation between social preference in three-chamber task and the proportion of CeA neurons that have an excitatory response to the social cup. The proportion of socially excited neurons is positively correlated with soc:obj zone ratio only for the GH on condition (Pearson correlation: r=0.6785, p=0.0445, N=9 mice). Bar and line graphs show mean ± SEM. *p<0.05.

Figure 7—source data 1 DRN^DAT-CeA:ChrimsonR three-chamber social:object ratio and social zone duration, as shown in Figure 7E.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig7-data1-v1.csv
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Figure 7—source data 2 CeA response strength to social and object stimuli, as shown in Figure 7G.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig7-data2-v1.csv
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Figure 7—source data 3 CeA response strength (change in auROC, social – object), as shown in Figure 7H.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig7-data3-v1.csv
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Figure 7—source data 4 CeA response overlap of social-encoding neurons, as shown in Figure 7I.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig7-data4-v1.csv
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Figure 7—source data 5 CeA response classification to social and object stimuli, as shown in Figure 7J.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig7-data5-v1.csv
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Figure 7—source data 6 Percentage of CeA neurons excited/inhibited by social stimulus, as shown in Figure 7K.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig7-data6-v1.csv
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Figure 7—source data 7 Percentage of CeA neurons excited/inhibited by object stimulus, as shown in Figure 7L.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig7-data7-v1.csv
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Figure 7—source data 8 Proportion of CeA neurons excited by social stimulus x social:object ratio, as shown in Figure 7M.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig7-data8-v1.csv
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We then performed microendoscopic epifluorescent calcium imaging during the three-chamber sociability task where mice freely explored a chamber containing a novel juvenile mouse and a novel object at opposite ends. Given that social isolation produces changes in long-term potentiation of synapses onto DRN^DAT neurons (Matthews et al., 2016), we were limited to a single manipulation of social isolation for each mouse. We hypothesized that stimulation of DRN^DAT inputs to CeA would mimic a loneliness-like state, consistent with our ChR2 manipulations with group-housed mice. Thus, we compared three conditions —group-housed without DRN^DAT stimulation (GH off), group-housed with DRN^DAT stimulation (GH on), and 24 hr socially isolated without DRN^DAT stimulation (SI off; Figure 7C, D) to allow for within-subjects comparisons.

In contrast to the photostimulation experiments in Figure 4, here we aimed to investigate the impact of DRN^DAT neuron stimulation on neural dynamics within the CeA without inducing robust behavioral changes that could introduce sensorimotor confounds to changes in neural activity due to stimulation. We successfully optimized viral expression and illumination parameters to minimize changes in social preference with DRN^DAT-CeA with ChrimsonR to prioritize comparison of the neural dynamics (Figure 7E) and also did not observe any behavioral effects of illumination in TdTomato-expressing mice (Figure 7—figure supplement 1J, K).

We then aligned the recorded CeA calcium traces with social cup and object cup interactions and found a striking diversity of neuronal responses to these stimuli under the three experimental conditions (Figure 7—figure supplement 2A, B). We next determined the response strength of individual CeA neurons to either stimulus under the three conditions (Figure 7F, G) using an area under ROC curve-based approach (Kingsbury et al., 2019; Li et al., 2017) to determine responsiveness of CeA neurons to social and object stimuli (Figure 7—figure supplement 1M). At a single-cell level, we did not observe significant changes in CeA response strength or proportion of neurons significantly responding to social or object stimuli across the three conditions (Figure 7G, H). However, we did find a trend indicating stronger responses toward social stimuli compared to object stimuli in the GH on condition compared to the GH off condition in mice expressing ChrimsonR (Figure 7J), but not TdTomato (Figure 7—figure supplement 1L) in DRN^DAT neurons. Importantly, in co-registered neurons, we found little overlap between CeA neurons excited by the social stimulus in both GH on and GH off conditions (Figure 7I), suggesting that DRN^DAT terminal stimulation may recruit separate ensembles of CeA neurons to represent social stimuli. Considering the variability in social preference behavior across mice and the diverse effects of photostimulation depending on the mouse’s social history, we next considered the responses of CeA neurons to social and object stimuli on an animal-by-animal basis. While we did not observe significant changes in the proportion of excitatory or inhibitory responses to social or object stimuli across the three conditions (Figure 7K, L), we did find a significant positive correlation between the proportion of socially excited CeA neurons and social preference in the GH on condition, but not in the GH off or SI off conditions (Figure 7M). Importantly, we do not observe a correlation between social preference and object-excited CeA neurons (Figure 7—figure supplement 1N) or socially inhibited CeA neurons (Figure 7—figure supplement 1O). This result may suggest that DRN^DAT input in the CeA in a behaviorally relevant task allows for a functional shift in its dynamics that enables it to predict the amount of social preference the mouse exhibits.

DRN^DAT-CeA photoinhibition blocks isolation-induced sociability

Finally, considering that the DRN^DAT-CeA projection is sufficient in promoting sociability, we next assessed whether activity in the DRN^DAT-CeA projection is necessary for the rebound in sociability that occurs following acute social isolation (Matthews et al., 2016). We injected an AAV enabling Cre-dependent expression of NpHR into the DRN of DAT::Cre male mice and implanted optic fibers over the BNST, CeA, or BLP (Figure 8A). We allowed 7 weeks for adequate terminal expression, after which we inhibited DRN^DAT terminals in the BNST, CeA, and BLP while mice performed the three-chamber sociability task (Figure 8B). Inhibition of DRN^DAT terminals in downstream regions while mice were group-housed did not change social preference (Figure 8—figure supplement 1A, B). However, inhibition of DRN^DAT terminals in CeA, but not BNST or BLP, blocked the rebound in sociability associated with acute social isolation (Figure 8C). Additionally, we found that optically inhibited changes in social preference in DRN^DAT-CeA mice were negatively correlated with social dominance (Figure 8D), suggesting that the DRN^DAT-CeA projection is necessary for the expression of isolation-induced social rebound in a rank-dependent manner.

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DRN^DAT-CeA photoinhibition blocks isolation-induced sociability.

(A) AAV₅-DIO-NpHR-eYFP or AAV₅-DIO-eYFP was injected into the DRN of DAT::Cre mice and optic fibers implanted over the BNST, CeA, or BLP to photoinhibit DRN^DAT terminals. (B) After >7 weeks for viral expression, mice were assayed on the three-chamber sociability assay with delivery of continuous yellow light for photoinhibition, once when group-housed and once following 24 hr of social isolation (2–3 weeks after the initial session). (C) Photoinhibition of DRN^DAT-BNST terminals in NpHR-expressing mice (DRN^DAT-BNST:NpHR) had no significant effect on time spent in the social zone relative to the object zone (DRN^DAT-BNST:NpHR: N=7 mice, DRN^DAT-BNST:eYFP: N=5 mice; ‘social:object ratio’; two-way RM ANOVA: light x group interaction, F_1,10=1.005, p=0.3397), but reduced social:object ratio for isolated DRN^DAT-CeA:NpHR mice compared to isolated DRN^DAT-CeA:eYFP mice (DRN^DAT-CeA:NpHR: N=20 mice, DRN^DAT-CeA:eYFP: N=12 mice; ‘social:object ratio’; two-way RM ANOVA: light x group interaction, F_1,30=4.909, p=0.0344; multiple comparisons test, DRN^DAT-CeA:NpHR^SI vs DRN^DAT-CeA:eYFP^SI adjusted **p=0.0017). In addition, terminal photoinhibition had no effect for DRN^DAT-BLP:NpHR mice (DRN^DAT-BLP:NpHR: N=6 mice, DRN^DAT-BLP:eYFP: N=8 mice; ‘social:object ratio’; two-way RM ANOVA: light x group interaction, F_1,12=3.346, p=0.0923). Inset bar graphs show the difference in social:object ratio in isolated and grouped conditions. A significant difference between NpHR^CeA and eYFP^CeA groups was observed (unpaired t-test: t₂₉=2.177, p=0.0377). (D) Scatter plots displaying relative dominance plotted against the change in social zone time (isolated-grouped), showing significant negative correlation in NpHR^CeA mice (Pearson’s correlation: r=−0.500, p=0.0414, N=20 mice). Bar and line graphs show mean ± SEM. *p<0.05, **p<0.01.

Figure 8—source data 1 DRN^DAT-ALL:NpHR three-chamber social:object ratio (GH on and SI on), as shown in Figure 8C.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig8-data1-v1.csv
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Figure 8—source data 2 DRN^DAT-ALL:NpHR three-chamber social:object ratio (SI – GH) x relative dominance, as shown in Figure 8D.: https://cdn.elifesciences.org/articles/105955/elife-105955-fig8-data2-v1.csv
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Discussion

Neural circuits that motivate social approach are essential in maintaining social connections and preventing isolation. Here we show that DRN^DAT neurons can exert a multi-faceted influence over behavior, with the pro-social effects mediated by the projection to the CeA, the avoidance effects mediated by the projection to the BLP, and the pro-exploratory effects mediated by the projection to the BNST. Our data suggest these effects are enabled via separable functional projections, dense collateralization, co-transmission, and precisely organized synaptic connectivity. Notably, our experiments were conducted in male mice; future work should investigate whether similar circuit mechanisms operate in females and explore the biological basis of sex-specific responses to social isolation (Yang et al., 2013; Zilkha et al., 2021). In addition, while terminal photostimulation allowed projection-specific manipulation, it may have inadvertently activated fibers of passage—a limitation that could be addressed in future studies using intersectional genetic strategies. Despite these caveats, these observed circuit features may facilitate a coordinated, but flexible, response in the presence of social stimuli that can be flexibly guided based on internal social homeostatic need state.

DRN^DAT circuit arrangement enables a broadly distributed, coordinated response

Our findings revealed several features of the DRN^DAT circuit which might facilitate a concerted response to novel social and non-social situations. Firstly, we observed dissociable roles for discrete downstream projections – a common motif of valence-encoding neural circuits (Tye, 2018). Biased recruitment of these ‘divergent paths’ Tye, 2018 to the BNST, CeA, and BLP by upstream inputs may serve to fine-tune the balance between social investigation and environmental exploration: facilitating behavioral flexibility with changing environmental conditions or internal state. Secondly, we demonstrate extensive collateralization of DRN^DAT neurons. In other populations, collateralization is proposed to aid temporal coordination of a multifaceted response: enabling synchronous activation of distributed regions (Rockland, 2018; Waselus et al., 2011). This feature may, therefore, facilitate coordinated recruitment of the BNST and CeA, allowing these regions to work in concert to promote social approach while also maintaining vigilance to salient environmental stimuli. Thirdly, we find precise synaptic organization in the DRN^DAT modulation of downstream neuronal activity that allows for qualitatively distinct response profiles of downstream targets. Combined with the spatially segregated downstream receptor expression pattern, this organization may allow DRN^DAT neurons to elicit broad, yet finely tuned, control over the pattern of neuronal activity, on multiple timescales—perhaps explaining the diverse behavior effects between the BNST- and CeA- projecting DRN^DAT populations, despite heavy collateralization.

Although we hypothesized that stimulating DRN^DAT inputs to the CeA in group-housed mice would mimic a state similar to that of isolation, we did not observe that the isolation OFF condition produced neural responses more similar to the group-housed ON condition. This suggests two possibilities: (1) that the photostimulation impacted neural activity beyond the endogenous dopamine innervation that may occur with social isolation because it is more potent of a change or (2) that the timing of endogenous dopamine innervation is different and is partially quenched upon exposure to a social agent. To completely understand the temporal dynamics of dopamine signaling with isolation and the firing of DRN^DAT neurons upon isolation, further experiments will require exploration of DRN^DAT stimulation parameter space, endogenous neural activity in the DRN^DAT-CeA circuit during social isolation, and the effects of DRN^DAT stimulation timing.

Separable projections mediate social behavior and valence

Our data support the hypothesis that separable DRN^DAT projections mediate distinct functional roles: a feature which has been previously observed in other neuronal circuits (e.g. Han et al., 2017; Kim et al., 2013; Kohl et al., 2018; Lammel et al., 2011; Namburi et al., 2015). The DRN^DAT circuit attributes we describe above may further enable this system to modulate other diverse forms of behavior (e.g. arousal Cho et al., 2017, fear/reward associations Lin et al., 2020; Groessl et al., 2018, and antinociception Li et al., 2016; Meyer et al., 2009; Yu et al., 2021). These could be mediated via other downstream projections and/or via these same projections under different environmental contexts, testing conditions, social histories, and/or internal states. Further work is required to determine how this system is able to exert a broad influence over multiple forms of behavior. Indeed, a recent study examined DRN^DAT projection to the nucleus accumbens and its role in promoting sociability (Choi et al., 2022), suggesting a parallel circuit to that described in the current study. Collectively, however, our data and others support a role for the DRN^DAT system in exerting a coordinated behavioral response to novel situations – both social and non-social.

The CeA has been implicated in mediating the response to threats – orchestrating defensive behavioral responses and autonomic changes via efferents to subcortical (Davis et al., 2010; Fadok et al., 2018; Gungor and Paré, 2016) and brainstem nuclei (Tovote et al., 2016). One possible interpretation, therefore, is that DRN^DAT input to the CeA suppresses fear-promoting neuronal ensembles in order to facilitate social approach. In the maintenance of social homeostasis, suppression of fear in the presence of social stimuli may represent an adaptive response – preventing salient social stimuli from being interpreted as a threat. Indeed, other need states, such as hunger, are associated with fear suppression and higher-risk behavior (Padilla et al., 2016), suggesting a conserved response to homeostatic imbalance (Matthews and Tye, 2019). However, the motivation to attend to social stimuli may also be driven by territorial defense (interacting with social rank), highlighting a need to further understand how internal states can play into the output of this system. A more comprehensive knowledge of the functional cell types modulated by DRN^DAT activity will facilitate our understanding of how this input can shape the downstream neuronal representation of social and non-social stimuli.

In contrast to the CeA, photoactivation of the DRN^DAT-BLP projection produced avoidance of the stimulation zone, suggesting an aversive state. This differs from the valence-independent role of VTA dopamine input to the greater BLA complex, wherein dopamine signaling gates synaptic plasticity for associative learning of both positive and negative valence (Tye et al., 2010) and responds to salient stimuli predicting both positive and negative outcomes (Lutas et al., 2019). However, DRN and VTA axonal fields differ within the BLA complex, with DRN^DAT terminals being more concentrated within the BLP, and VTA^DAT inputs traversing the LA, BLA, and intercalated cells more densely.

While there have been seemingly contradictory reports on the effect of dopamine on excitability in the BLA (Tye et al., 2010; Bissière et al., 2003; Lutas et al., 2019), our observations using photostimulation of DRN^DAT terminals (in short phasic bursts) are consistent with in vivo extracellular recordings combined with electrical stimulation of the midbrain (Rosenkranz and Grace, 1999). One unifying hypothesis is that dopamine induces an indirect GABA-mediated suppression of pyramidal neurons, which may attenuate their response to weak inputs, while directly exciting pyramidal neurons to augment their response to large inputs (Kröner et al., 2005; Rosenkranz and Grace, 1999). In this way, amygdala dopamine may underlie a similar role to cortical dopamine (Vander Weele et al., 2018; Gulledge and Jaffe, 2001) – enhancing signal-to-noise ratio to facilitate behavioral responses to salient stimuli (Vander Weele et al., 2018).

A similar complexity surrounds dopamine’s effects in the BNST. Photostimulation of DRN^DA-BNST projections has been shown to have antinociceptive effects on male mice upon formalin injection into the paw, whereas females do not show this antinociceptive effect and instead display contextual hyperlocomotion (Yu et al., 2021). Given that our study does not include females, there are likely behavioral and physiological sex differences that beg further investigation. Additionally, prior studies using optogenetic stimulation (Li et al., 2016; Yu et al., 2021) or exogenously applied dopamine (Krawczyk et al., 2011; Maracle et al., 2019) report a wide range of outcomes on BNST excitability, including a higher frequency of inhibitory responses that we observed. Notably, one study found that higher doses of exogenously applied dopamine decreased the amplitude of GABA_A-IPSC in ovBNST neurons (Krawczyk et al., 2011), potentially consistent with our results. Interestingly, animals given intermittent access to sucrose displayed a significant increase in GABA_A-IPSCs in ovBNST neurons following dopamine application (Maracle et al., 2019), raising the possibility that experience-dependent factors shape dopamine sensitivity in the BNST, paralleling how dopaminergic modulation in the cortex may be state-dependent (Vander Weele et al., 2018). Considering that our ex vivo recordings were performed in group-housed mice, future work should test whether social isolation alters ovBNST responses to dopamine, providing a framework for understanding how environmental states influence dopaminergic neuromodulatory tone.

Multi-transmitter phenotype of DRN^DAT neurons may permit modulation on different timescales

DRN^DAT neurons possess an impressive repertoire of signaling molecules: alongside dopamine and glutamate subsets of DRN^DAT neuron express VIP and NPW (Dougalis et al., 2012; Huang et al., 2019; Motoike et al., 2016). While there is some partial segregation of VIP- and NPW-expressing neurons (Huang et al., 2019), our receptor expression analyses suggest that these neuropeptides converge on the same neurons in the BNST and CeA. This co-localization is intriguing, given that Vipr2 is typically coupled to the excitatory G_s-protein (White et al., 2010), while Npbwr1 is coupled to the inhibitory G_i-protein (Nagata-Kuroiwa et al., 2011; Tanaka et al., 2003). Therefore, signaling through these receptors may exert opposing actions on downstream cells. Recruitment of neuropeptidergic signaling pathways may support slower, sustained downstream modulation, for example, in hunger-mediating hypothalamic Agouti-Related Peptide (AgRP) neurons, neuropeptide co-release is essential for sustaining feeding behavior (Chen et al., 2019). Therefore, a delayed, persistent neuropeptide-mediated signal might enable downstream modulation to outlive phasic DRN^DAT activity: promoting behavioral adjustments over longer timescales.

While the functional role of these neuropeptides remains to be elucidated, studies on knockout mice suggest a role for NPW in social behavior and stress responding (Nagata-Kuroiwa et al., 2011; Motoike et al., 2016). Furthermore, in humans with a single-nucleotide polymorphism (SNP) of the NPBWR1 gene (which impairs receptor function), the perception of fearful/angry faces was more positive and less submissive (Watanabe et al., 2012), suggesting a possible role for NPW signaling in interpreting social signals. Similarly, the function of DRN VIP +neurons has received little attention in rodent models, but there has been more focus on the role of VIP in avian social behavior (Kingsbury and Wilson, 2016). Of particular interest, in the rostral arcopallium (a homolog of mammalian amygdala Reiner et al., 2004), VIP binding density is elevated in birds during seasonal flocking (Wilson et al., 2016). This suggests that elevated VIP receptor expression may encourage affiliative social grouping behavior in birds (Wilson et al., 2016). Thus, NPW and VIP may act in concert with fast glutamate-mediated and slow dopamine-mediated neurotransmission in the central extended amygdala to modulate behavior on different timescales.

Together, these findings suggest that NPW and VIP may act in concert with fast glutamate and slower dopamine signaling to shape social behavior across multiple timescales within the extended amygdala. Although we observed relatively low expression of Drd1 in the BNST and CeA compared to Vipr2 and Npbwr1, it remains possible that dopamine’s effects are mediated by other receptors, such as Drd3, Drd4, and Drd5—warranting further investigation.

Conclusion

Together, these findings reveal that DRN^DAT projections exhibit substantial functional specialization, with anatomically distinct pathways modulating different facets of behavior. The DRN^DAT-CeA projection promotes sociability, DRN^DAT-BLP input drives avoidance, and DRN^DAT-BNST enhances vigilant exploration, highlighting the diverse roles of this neural circuit in coordinating adaptive responses to social and environmental contexts. These findings uncover a circuit mechanism through which DRN^DAT projections orchestrate distinct behavioral features of a loneliness-like state, providing a framework for understanding how neuromodulatory systems guide complex social and emotional behaviors and suggesting potential targets for therapeutic intervention in affective disorders.

Materials and methods

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Strain, strain background (Mus musculus, male)	DAT^IREScre (B6.SJL-Slc6a3^{tm1.1(cre)Bkmn}/J)	Jackson Laboratory	IMSR_JAX: 000664
Strain, strain background (M. musculus, male)	C57BL/6 J	Charles River Laboratories	Strain code 631
Strain, strain background (AAV)	AAV5-EF1α-DIO-ChR2-eYFP	University of North Carolina Vector Core		https://www.med.unc.edu/genetherapy/vectorcore/in-stock-aav-vectors/deisseroth/
Strain, strain background (AAV)	AAV5-EF1α-DIO-eYFP	University of North Carolina Vector Core		https://www.med.unc.edu/genetherapy/vectorcore/in-stock-aav-vectors/deisseroth/
Strain, strain background (AAV)	AAV9-Syn-ChrimsonR-tdTomato	University of North Carolina Vector Core		https://www.med.unc.edu/genetherapy/vectorcore/in-stock-aav-vectors/deisseroth/
Strain, strain background (AAV)	HSV-LS1L-mCherry-IRES-flpo	Viral Gene Transfer Core Facility at MIT		https://biology.mit.edu/faculty-and-research/core-facilities/
Strain, strain background (AAV)	AAV5-EF1α-DIO-eNpHR3.0-eYFP	Addgene	RRID:Addgene_26966
Strain, strain background (AAV)	AAV1-syn-jGCaMP7f	Addgene	RRID:Addgene_104488
Strain, strain background (AAV)	AAV1-CAG-TdTomato	UPenn vector core		https://www.med.upenn.edu/carot/aav-core.html
Chemical compound	CTB-555	ThermoFisher	C34776
Chemical compound	CTB-647	ThermoFisher	C34778
Chemical compound	DAPI	Sigma-Aldrich	D9542	1:50,000
Chemical compound	PVA-DABCO	Sigma-Aldrich	10981
Chemical compound	Normal Donkey Serum	Jackson Immunoresearch	RRID:AB_2337258
Chemical compound	CF405-conjugated streptavidin	Biotium	29032	1:1000
Chemical compound, drug	CF633-conjugated streptavidin	Biotium	29037	1:1000
Chemical compound	RNAScope Multiplex Fluorescent Reagent Kit V2	ACDBio	323110
Chemical compound	RNAScope Multiplex Detection Reagents	ACDBio	323110
Chemical compound	RNAScope Drd1a target probe	ACDBio	406491-C1
Chemical compound	RNAScope Drd2 target probe	ACDBio	406501-C3
Chemical compound	RNAScope Npbwr1 target probe	ACDBio	547181-C1
Chemical compound	RNAScope Vipr2 target probe	ACDBio	465391-C1
Chemical compound	TSA fluorophore (green)	PerkinElmer	Fluorescein
Chemical compound	TSA fluorophore (red)	PerkinElmer	Cyanine 3
Chemical compound	TSA fluorophore (far red)	PerkinElmer	Cyanine 5
Antibody	Anti-Tyrosine Hydroxylase Antibody	Millipore	RRID:AB_570923	1:1000
Antibody	Alexa Fluor 488 AffiniPure Donkey Anti-Chicken IgY (IgG) (H+L)	Jackson ImmunoResearch	RRID:AB_2340375	1:1000
Antibody	Alexa Fluor 647-AffiniPure Donkey Anti-Chicken IgY (IgG) (H+L)	Jackson ImmunoResearch	RRID:AB_2340379	1:1000
Software	Fluoview software version 4.0	Olympus	RRID:SCR_014215
Software	FIJI	ImageJ	RRID:SCR_003070
Software	CellProfiler	CellProfiler	RRID:SCR_007358
Software	Adobe Photoshop CC	Adobe	RRID:SCR_014199
Software	MATLAB	Mathworks	RRID:SCR_001622
Software	Adobe Illustrator	Adobe	RRID:SCR_010279
Software	Ethovision XT	Noldus	RRID:SCR_000441
Software	pClamp 10.4 software	Molecular Devices	RRID:SCR_011323
Software	GraphPad Prism 8	Graphpad	RRID:SCR_002798
Other	Optic fiber	Thor Labs	TS1843490
Other	Ferrules	Kientec Systems	FSS-LC-330
Other	Small animal stereotaxic frame	David Kopf Instruments	Model 942
Other	0.10 mL Microsyringe	World Precision Instruments (NANOFIL-NF33BL-2)	RRID:SCR_008593
Other	Microsyringe Pump UMP3 and Controller Micro4	World Precision Instruments (UMP3-3)	RRID:SCR_008593
Other	Peristaltic pump for ex vivo recordings	Minipuls 3 Gilson	F155001
Other	Pulse generator	A.M.P.I. Master-8	RRID:SCR_018889
Other	Cryostat	Leica biosystems CM3050 S	RRID:SCR_020214
Other	HM430 Microtome	Thermo Fisher Scientific	RRID:SCR_020020
Other	Vibrating blade vibratome	Leica Biosystems VT1200	RRID:SCR_018453
Other	Confocal Laser-Scanning microscope	Olympus FV1000	RRID:SCR_020337
Other	Diode Blue 473 nm Laser	OptoEngine LLC	MBL-III-473/1–100 mW
Other	Horizontal puller for glass microelectrodes for ex vivo recordings	Sutter P-1000	RRID:SCR_021042
Other	Multiclamp amplifier for ex vivo recordings	Molecular Devices 700B	RRID:SCR_018455
Other	Microscope for ex vivo recordings	Scientifica	RRID:SCR_021035
Other	Microscope for in vivo calcium imaging recordings	Inscopix nVoke System	RRID:SCR_023028

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DRNDAT and VTADAT afferents target distinct downstream regions.

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DRNDAT-BLP (but not DRNDAT-BNST or DRNDAT-CeA) photostimulation promotes place avoidance.

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DRNDAT-BNST (but not DRNDAT-CeA or DRNDAT-BLP) photostimulation promotes non-social exploratory behavior.

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DRNDAT-CeA (but not DRNDAT-BNST or DRNDAT-BLP) photostimulation promotes sociability in a rank-dependent manner.

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Spatial segregation of dopamine and neuropeptide receptor populations within DRNDAT terminal fields.

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DRNDAT input distinctly influences downstream activity in each downstream target.

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Simultaneous calcium imaging of CeA neurons and optogenetic stimulation of DRNDAT terminals in CeA.

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DRNDAT-CeA photoinhibition blocks isolation-induced sociability.

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Author details

Christopher R Lee

Contribution

DRN^DAT and VTA^DAT afferents target distinct downstream regions.

DRN^DAT-BLP (but not DRN^DAT-BNST or DRN^DAT-CeA) photostimulation promotes place avoidance.

DRN^DAT-BNST (but not DRN^DAT-CeA or DRN^DAT-BLP) photostimulation promotes non-social exploratory behavior.

DRN^DAT-CeA (but not DRN^DAT-BNST or DRN^DAT-BLP) photostimulation promotes sociability in a rank-dependent manner.

Spatial segregation of dopamine and neuropeptide receptor populations within DRN^DAT terminal fields.

DRN^DAT input distinctly influences downstream activity in each downstream target.

Simultaneous calcium imaging of CeA neurons and optogenetic stimulation of DRN^DAT terminals in CeA.

DRN^DAT-CeA photoinhibition blocks isolation-induced sociability.