CoChat
Sign Up
Cited by 402Neurotransmitter Receptor Influence on BehaviorNeuroendocrine regulation and behaviorNeuroscience and Neuropharmacology Research

The DOI matches its Crossref record; 73 of the 73 references it cites were found in a registry or an index; none of the 62 sources its claims cite is checked for support yet.

Dorsal Raphe Dopamine Neurons Represent the Experience of Social Isolation

Open access
Matthews, Gillian A., Nieh, Edward H., Vander Weele, Caitlin M., Halbert, Sarah A., Pradhan, Roma V., Yosafat, Ariella S., Glober, Gordon F., Izadmehr, Ehsan M., Thomas, Rain E., Lacy, Gabrielle D., Wildes, Craig P., Ungless, Mark A., Tye, Kay M.·2016·Cell
DOI 10.1016/j.cell.2015.12.040PMC PMC4752823
Open this paper in CoChat — free accountOpen PDF

Abstract

The motivation to seek social contact may arise from either positive or negative emotional states, as social interaction can be rewarding and social isolation can be aversive. While ventral tegmental area (VTA) dopamine (DA) neurons may mediate social reward, a cellular substrate for the negative affective state of loneliness has remained elusive. Here, we identify a functional role for DA neurons in the dorsal raphe nucleus (DRN), in which we observe synaptic changes following acute social isolation. DRN DA neurons show increased activity upon social contact following isolation, revealed by in vivo calcium imaging. Optogenetic activation of DRN DA neurons increases social preference but causes place avoidance. Furthermore, these neurons are necessary for promoting rebound sociability following an acute period of isolation. Finally, the degree to which these neurons modulate behavior is predicted by social rank, together supporting a role for DRN dopamine neurons in mediating a loneliness-like state. PAPERCLIP.

Overview

AI snapshot

Written from the paper’s full text.

TL;DR

Dorsal raphe nucleus (DRN) dopamine neurons undergo synaptic potentiation after 24 hr social isolation, increase activity upon first social contact, and are sufficient and necessary for isolation-induced sociability. Their activation promotes social preference but is aversive without a social target, suggesting they represent a loneliness-like state modulated by social rank.

Key findings

  • 24 hr social isolation increased the AMPAR/NMDAR ratio in DRN DA neurons but not VTA DA neurons; isolated mice also showed a greater AMPAR rectification index and NASPM sensitivity, suggesting a relative increase in GluR2-lacking AMPARs.
  • In vivo fiber photometry showed that socially isolated mice had a significantly greater DRN DA calcium signal in response to first contact with a juvenile mouse than group-housed mice, and greater than to a novel object.
  • Optogenetic activation of DRN DA neurons increased social preference in the three-chamber task but did not support ICSS and produced real-time place avoidance and conditioned place avoidance; photoinhibition reduced social preference after isolation but not in group-housed mice.
  • Social rank predicted behavioral effects: dominance positively correlated with activation-induced social preference and negatively with stimulation-zone preference, while photoinhibition reduced social preference more in dominant mice after isolation. DRN DA neurons also released DA and glutamate in dlBNST and CeA; EPSCs were elicited in 25/30 dlBNST neurons and 17/21 CeA neurons, and 77.7% of eYFP+ DRN neurons in TH::Cre mice and 79.8% in DAT::IRES-Cre mice co-labeled with TH.

Methods

  • Ex vivo whole-cell patch-clamp electrophysiology in brain slices (220 μm) from male TH-GFP or Pitx3-GFP mice, measuring AMPAR/NMDAR ratio, rectification index, paired-pulse ratio, and NASPM sensitivity after 24 hr social isolation versus group housing.
  • Fiber photometry in TH::Cre mice with AAV5-CAG-FLEX-GCaMP6m targeted to DRN DA neurons; GCaMP6m fluorescence was recorded in the home cage before and after introduction of a juvenile mouse or novel object.
  • Optogenetic and behavioral experiments in TH::Cre and DAT::Cre mice with AAV5-DIO-ChR2-eYFP, AAV5-DIO-NpHR3.0-eYFP, or AAV5-DIO-eYFP in DRN; assays included three-chamber sociability, ICSS, real-time place avoidance, conditioned place preference, in vivo fast-scan cyclic voltammetry, and immunohistochemistry.

Limitations

  • The paper states that “loneliness” per se is difficult to directly test in mice; it also states that it remains undetermined whether underlying neural differences cause social rank and/or whether social rank itself changes neuronal properties, and that CeA DA transients were inconsistent and did not adequately resolve as DA.
  • Evident from the text: ex vivo electrophysiology used male mice, and dominance measures are described for males, so sex differences are not addressed.
  • Evident from the text: social-rank effects are correlational, and the “loneliness-like state” interpretation is inferred from mouse behavior rather than directly measured subjective experience.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1016/j.cell.2015.12.040

Cites

73 of 73 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Figures

Graphical abstract
Graphical abstract CC BY · PubMed Central
Figure 1 24 Hours of Social Isolation Induces Synaptic Potentiation onto DRN DA Neurons (A) AMPAR/NMDAR ratios recorded from VTA DA neurons in mice socially isolated for 24 hr (n = 12) were not significantly different from group-housed mice (n = 8; unpaired t test: t18 = 0.73, p = 0.47). (B) Low- (upper panel) and high-magnification (lower panels) confocal images of the DRN from a TH-GFP mouse showing GFP-expressing (green) and post hoc immunohistochemically verified TH-expressing (red) DA neurons with white arrows indicating co-labeled neurons. (C) AMPAR/NMDAR ratios recorded from DRN DA neurons in mice socially isolated for 24 hr, either in a familiar cage or a novel cage (familiar isolated or novel isolated, respectively), were significantly greater than group-housed mice in familiar or novel cages (one-way ANOVA: F3,47 = 5.910, ∗∗p = 0.0017; Newman-Keuls post hoc tests: ∗p < 0.05, ∗∗p < 0.01; n = 19 naive, 17 familiar isolated, 9 novel grouped, and 6 novel isolated). Scale bars, 20 pA, 20 ms. (D) The AMPAR rectification index in DRN DA neurons was significantly greater in socially isolated mice, relative to naive mice (unpaired t test: t21 = 2.417, ∗p = 0.0248, n = 9 naive, 14 isolated). (E) Normalized AMPAR-mediated EPSC amplitude during bath application of NASPM, and representative averaged EPSCs from a naive and socially isolated mouse (inset shows % change in EPSC amplitude following NASPM, relative to baseline). NASPM significantly reduced EPSC amplitude in socially isolated mice (n = 7), relative to naive mice (n = 8; unpaired t test: t13 = 2.853, ∗p = 0.0136). Scale bars, 10 pA, 10 ms. (F) Proposed model of AMPARs at synapses onto DRN DA neurons in group-housed mice and following social isolation. Data are represented as mean ± SEM. See also Figure S1.
Figure 1 24 Hours of Social Isolation Induces Synaptic Potentiation onto DRN DA Neurons (A) AMPAR/NMDAR ratios recorded from VTA DA neurons in mice socially isolated for 24 hr (n = 12) were not significantly different from group-housed mice (n = 8; unpaired t test: t18 = 0.73, p = 0.47). (B) Low- (upper panel) and high-magnification (lower panels) confocal images of the DRN from a TH-GFP mouse showing GFP-expressing (green) and post hoc immunohistochemically verified TH-expressing (red) DA neurons with white arrows indicating co-labeled neurons. (C) AMPAR/NMDAR ratios recorded from DRN DA neurons in mice socially isolated for 24 hr, either in a familiar cage or a novel cage (familiar isolated or novel isolated, respectively), were significantly greater than group-housed mice in familiar or novel cages (one-way ANOVA: F3,47 = 5.910, ∗∗p = 0.0017; Newman-Keuls post hoc tests: ∗p < 0.05, ∗∗p < 0.01; n = 19 naive, 17 familiar isolated, 9 novel grouped, and 6 novel isolated). Scale bars, 20 pA, 20 ms. (D) The AMPAR rectification index in DRN DA neurons was significantly greater in socially isolated mice, relative to naive mice (unpaired t test: t21 = 2.417, ∗p = 0.0248, n = 9 naive, 14 isolated). (E) Normalized AMPAR-mediated EPSC amplitude during bath application of NASPM, and representative averaged EPSCs from a naive and socially isolated mouse (inset shows % change in EPSC amplitude following NASPM, relative to baseline). NASPM significantly reduced EPSC amplitude in socially isolated mice (n = 7), relative to naive mice (n = 8; unpaired t test: t13 = 2.853, ∗p = 0.0136). Scale bars, 10 pA, 10 ms. (F) Proposed model of AMPARs at synapses onto DRN DA neurons in group-housed mice and following social isolation. Data are represented as mean ± SEM. See also Figure S1.CC BY · PubMed Central
Figure 2 DRN DA Neurons Track Initial Social Contact Following Social Isolation (A) Schematic for recording activity of GCaMP6m-expressing neurons. (B) Image showing Cre-dependent expression of GCaMP6m in the DRN of a TH::Cre mouse, with optic fiber placement indicated. (C) Representative traces of bulk fluorescence signal from DRN DA neurons, with shaded areas indicating interaction bouts. Mice were recorded under three conditions: group-housed mice presented with a juvenile mouse (gray), socially isolated mice presented with a juvenile mouse (lilac), or socially isolated mice presented with a novel object (teal). (D) Heat maps showing the individual Z scores in response to the first interaction bout for each animal under each condition. (E) Population Z score plots showing the averaged response to the first interaction bout. (F) DRN DA neurons in socially isolated mice showed a significantly greater increase in activity upon first contact with the juvenile mouse, compared with group-housed mice or response to a novel object (n = 9; one-way ANOVA: F2,16 = 4.978, ∗p = 0.0208; Bonferroni post hoc analysis: ∗p < 0.05 for both comparisons). Data are represented as mean ± SEM. See also Figure S2 and Movie S1.
Figure 2 DRN DA Neurons Track Initial Social Contact Following Social Isolation (A) Schematic for recording activity of GCaMP6m-expressing neurons. (B) Image showing Cre-dependent expression of GCaMP6m in the DRN of a TH::Cre mouse, with optic fiber placement indicated. (C) Representative traces of bulk fluorescence signal from DRN DA neurons, with shaded areas indicating interaction bouts. Mice were recorded under three conditions: group-housed mice presented with a juvenile mouse (gray), socially isolated mice presented with a juvenile mouse (lilac), or socially isolated mice presented with a novel object (teal). (D) Heat maps showing the individual Z scores in response to the first interaction bout for each animal under each condition. (E) Population Z score plots showing the averaged response to the first interaction bout. (F) DRN DA neurons in socially isolated mice showed a significantly greater increase in activity upon first contact with the juvenile mouse, compared with group-housed mice or response to a novel object (n = 9; one-way ANOVA: F2,16 = 4.978, ∗p = 0.0208; Bonferroni post hoc analysis: ∗p < 0.05 for both comparisons). Data are represented as mean ± SEM. See also Figure S2 and Movie S1.CC BY · PubMed Central
Figure 3 Photostimulation of DRN DA Neurons Elicits DA Release in the BNST and CeA (A) AAV5-DIO-ChR2-eYFP was injected into the DRN of TH::Cre mice to facilitate (B) ChR2 (green) expression in TH+ (red) DRN neurons. White arrows indicate selected co-labeled neurons. (C) Example of a biocytin-filled, ChR2-expressing, DRN neuron recorded using ex vivo electrophysiology. (D) ChR2 was activated using eight 5 ms pulses of blue light delivered every 5 s, which elicited a train of action potentials in the ChR2-expressing neuron. (E) Sagittal brain sections showing dense terminal expression in the BNST and (F) CeA following AAV5-DIO-eYFP injection into the DRN of a TH::Cre mouse. (G) In vivo FSCV was performed in anesthetized TH::Cre mice following Cre-dependent expression of ChR2 in the DRN. Example color plots and average traces (± SEM) from the BNST (n = 5 mice; 7 recording sites) and (H) CeA (n = 4 mice; 5 recording sites) showing DA release evoked by 150 5 ms pulses of blue light delivered at 30 Hz (left panels) and a representative trace showing eight 5 ms pulses delivered every 5 s (right panels). Eapp, applied potential. Insets show cyclic voltammograms from representative color plots. See also Figures S2 and S3.
Figure 3 Photostimulation of DRN DA Neurons Elicits DA Release in the BNST and CeA (A) AAV5-DIO-ChR2-eYFP was injected into the DRN of TH::Cre mice to facilitate (B) ChR2 (green) expression in TH+ (red) DRN neurons. White arrows indicate selected co-labeled neurons. (C) Example of a biocytin-filled, ChR2-expressing, DRN neuron recorded using ex vivo electrophysiology. (D) ChR2 was activated using eight 5 ms pulses of blue light delivered every 5 s, which elicited a train of action potentials in the ChR2-expressing neuron. (E) Sagittal brain sections showing dense terminal expression in the BNST and (F) CeA following AAV5-DIO-eYFP injection into the DRN of a TH::Cre mouse. (G) In vivo FSCV was performed in anesthetized TH::Cre mice following Cre-dependent expression of ChR2 in the DRN. Example color plots and average traces (± SEM) from the BNST (n = 5 mice; 7 recording sites) and (H) CeA (n = 4 mice; 5 recording sites) showing DA release evoked by 150 5 ms pulses of blue light delivered at 30 Hz (left panels) and a representative trace showing eight 5 ms pulses delivered every 5 s (right panels). Eapp, applied potential. Insets show cyclic voltammograms from representative color plots. See also Figures S2 and S3.CC BY · PubMed Central
Figure 4 Optical Stimulation of DRN DA Neurons Elicits Monosynaptic Glutamate Release in the BNST and CeA (A) Ex vivo electrophysiology was performed in the BNST and (B) CeA following Cre-dependent expression of ChR2 in the DRN of TH::Cre and DAT::Cre mice. (C and D) Optical stimulation of ChR2-expressing terminals with a 5 ms blue light pulse elicited a short-latency, fast EPSC (measured in voltage-clamp at −70 mV), which persisted in the presence of TTX/4AP, but no detectable short-latency IPSC (measured at 0 mV). Scale bars, 10 pA, 10 ms. (E) The proportion of recorded neurons in the BNST and (F) CeA that responded with an EPSC (in the absence of TTX/4AP) and the amplitude and latency of each. (G and H) Individual traces showing the long-latency IPSCs elicited by successive 5 ms pulses of blue light, delivered every 20 s. Insets show average IPSC latency; pie charts show the proportion of cells which responded with an IPSC. Scale bars, 50 pA, 10 ms. (I and J) Confocal images showing eYFP-expressing neurons (green) in the DRN with post hoc immunohistochemistry for TH (red), following Cre-dependent expression of eYFP, in VGLUT2::Cre and (K and L) VGAT::Cre mice. White arrows indicate selected co-labeled cells. A significantly greater proportion of TH+ neurons co-expressed VGLUT2 (n = 119/582 neurons) compared with VGAT (n = 1/577 neurons; Chi-square = 128.30, p < 0.0001). Data are represented as mean ± SEM. See also Figure S4.
Figure 4 Optical Stimulation of DRN DA Neurons Elicits Monosynaptic Glutamate Release in the BNST and CeA (A) Ex vivo electrophysiology was performed in the BNST and (B) CeA following Cre-dependent expression of ChR2 in the DRN of TH::Cre and DAT::Cre mice. (C and D) Optical stimulation of ChR2-expressing terminals with a 5 ms blue light pulse elicited a short-latency, fast EPSC (measured in voltage-clamp at −70 mV), which persisted in the presence of TTX/4AP, but no detectable short-latency IPSC (measured at 0 mV). Scale bars, 10 pA, 10 ms. (E) The proportion of recorded neurons in the BNST and (F) CeA that responded with an EPSC (in the absence of TTX/4AP) and the amplitude and latency of each. (G and H) Individual traces showing the long-latency IPSCs elicited by successive 5 ms pulses of blue light, delivered every 20 s. Insets show average IPSC latency; pie charts show the proportion of cells which responded with an IPSC. Scale bars, 50 pA, 10 ms. (I and J) Confocal images showing eYFP-expressing neurons (green) in the DRN with post hoc immunohistochemistry for TH (red), following Cre-dependent expression of eYFP, in VGLUT2::Cre and (K and L) VGAT::Cre mice. White arrows indicate selected co-labeled cells. A significantly greater proportion of TH+ neurons co-expressed VGLUT2 (n = 119/582 neurons) compared with VGAT (n = 1/577 neurons; Chi-square = 128.30, p < 0.0001). Data are represented as mean ± SEM. See also Figure S4.CC BY · PubMed Central
Figure 5 Optical Activation of DRN DA Neurons Elicits Social Preference and Place Avoidance (A) TH::Cre mice were tested for social preference in the three-chamber sociability task. (B) Representative spatial heat maps showing the location of a ChR2-expressing mouse. (C) Time spent by ChR2- and eYFP-expressing mice in the social zone across the first 5 min of the task, with and without photostimulation (inset shows total time). ChR2-expressing, but not eYFP-expressing, mice showed a significant increase in the total time spent in the social zone, relative to the non-social zone, when receiving blue light stimulation (ChR2, inset, paired t test: t10 = 3.297, ∗∗p = 0.0081, n = 11; eYFP: t10 = 0.0100, p = 0.9922; n = 11) and (D) a significant increase in the social:non-social ratio (ChR2, paired t test: t10 = 3.843, ∗∗p = 0.0032, n = 11; eYFP: t10 = 0.1847, p = 0.8572, n = 11). (E) Cumulative activity graph of nose pokes made by a ChR2-expressing mouse at the inactive and active (light-paired) ports during an ICSS task. (F) Optical stimulation of DRN DA neurons did not support ICSS, as revealed by the number of nose pokes made into the inactive and active ports by ChR2- (active versus inactive, paired t test: t4 = 0.0811, p = 0.9393, n = 5) and eYFP-expressing mice (active versus inactive, paired t test: t6 = 0.732, p = 0.4917, n = 7). (G) CPP paradigm and (H) representative track from a ChR2-expressing mouse during the first 10 min of the test session. (I) Graph showing % time spent in the previously light-paired zone (inset shows first 10 min). ChR2-expressing mice showed significant avoidance of the previously light-paired zone, relative to eYFP mice, as shown by the % time spent in this zone (unpaired t test: t10 = 2.393, ∗p = 0.0378, n = 6 ChR2, 6 eYFP) and (J) the difference between the time spent in the previously light-paired and unpaired zones (unpaired t test: t10 = 2.241, ∗p = 0.0489, n = 6 ChR2, 6 eYFP). Data are represented as mean ± SEM. See also Figure S5.
Figure 5 Optical Activation of DRN DA Neurons Elicits Social Preference and Place Avoidance (A) TH::Cre mice were tested for social preference in the three-chamber sociability task. (B) Representative spatial heat maps showing the location of a ChR2-expressing mouse. (C) Time spent by ChR2- and eYFP-expressing mice in the social zone across the first 5 min of the task, with and without photostimulation (inset shows total time). ChR2-expressing, but not eYFP-expressing, mice showed a significant increase in the total time spent in the social zone, relative to the non-social zone, when receiving blue light stimulation (ChR2, inset, paired t test: t10 = 3.297, ∗∗p = 0.0081, n = 11; eYFP: t10 = 0.0100, p = 0.9922; n = 11) and (D) a significant increase in the social:non-social ratio (ChR2, paired t test: t10 = 3.843, ∗∗p = 0.0032, n = 11; eYFP: t10 = 0.1847, p = 0.8572, n = 11). (E) Cumulative activity graph of nose pokes made by a ChR2-expressing mouse at the inactive and active (light-paired) ports during an ICSS task. (F) Optical stimulation of DRN DA neurons did not support ICSS, as revealed by the number of nose pokes made into the inactive and active ports by ChR2- (active versus inactive, paired t test: t4 = 0.0811, p = 0.9393, n = 5) and eYFP-expressing mice (active versus inactive, paired t test: t6 = 0.732, p = 0.4917, n = 7). (G) CPP paradigm and (H) representative track from a ChR2-expressing mouse during the first 10 min of the test session. (I) Graph showing % time spent in the previously light-paired zone (inset shows first 10 min). ChR2-expressing mice showed significant avoidance of the previously light-paired zone, relative to eYFP mice, as shown by the % time spent in this zone (unpaired t test: t10 = 2.393, ∗p = 0.0378, n = 6 ChR2, 6 eYFP) and (J) the difference between the time spent in the previously light-paired and unpaired zones (unpaired t test: t10 = 2.241, ∗p = 0.0489, n = 6 ChR2, 6 eYFP). Data are represented as mean ± SEM. See also Figure S5.CC BY · PubMed Central
Figure 6 Optical Inhibition of DRN DA Neurons Reduces Social Preference Only Following Isolation (A) Group-housed TH::Cre mice expressing NpHR (upper panels) or eYFP (lower panels) in the DRN, in a Cre-dependent manner, were assessed in the three-chamber sociability task. Time spent in the social zone across the first 5 min of the task (inset shows total time) and (B) the social:non-social ratio. Optical inhibition did not produce a detectable difference in the total time spent in the social zone (NpHR, inset, paired t test: t12 = 0.1778 p = 0.8619, n = 13; eYFP: t9 = 0.1788, p = 0.8621, n = 10) or the social:non-social ratio (NpHR, paired t test: t12 = 0.2414, p = 0.8133, n = 13; eYFP: t9 = 1.293, p = 0.2282, n = 10). (C) Mice were isolated from their cagemates for 24 hr and then tested for sociability with optical inhibition. Time spent in the social zone by NpHR- and eYFP-expressing mice across the first 5 min (inset shows total time) and social:non-social ratio following isolation. NpHR-expressing mice spent a significantly lower proportion of time in the social zone after 24 hr of isolation compared with eYFP-expressing mice (social:non-social ratio, unpaired t test: t16 = 2.236, ∗p = 0.0400, n = 7 eYFP, 11 NpHR). (D) Representative spatial heat maps showing the location of an NpHR- (upper panels) and eYFP-expressing mouse (lower panels) in the first 5 min of the task, when group-housed and following 24 hr of isolation. (E) Time spent in the social zone and social:non-social ratio of mice tested with photoinhibition while group-housed and following social isolation. Photoinhibition in NpHR-expressing mice resulted in a significant reduction in time spent in the social zone (paired t test: t10 = 2.740, ∗p = 0.0208, n = 11) and social:non-social ratio (paired t test: t10 = 2.239, p = ∗0.0491, n = 11) following social isolation, compared to when group-housed. Data are represented as mean ± SEM. See also Figure S6.
Figure 6 Optical Inhibition of DRN DA Neurons Reduces Social Preference Only Following Isolation (A) Group-housed TH::Cre mice expressing NpHR (upper panels) or eYFP (lower panels) in the DRN, in a Cre-dependent manner, were assessed in the three-chamber sociability task. Time spent in the social zone across the first 5 min of the task (inset shows total time) and (B) the social:non-social ratio. Optical inhibition did not produce a detectable difference in the total time spent in the social zone (NpHR, inset, paired t test: t12 = 0.1778 p = 0.8619, n = 13; eYFP: t9 = 0.1788, p = 0.8621, n = 10) or the social:non-social ratio (NpHR, paired t test: t12 = 0.2414, p = 0.8133, n = 13; eYFP: t9 = 1.293, p = 0.2282, n = 10). (C) Mice were isolated from their cagemates for 24 hr and then tested for sociability with optical inhibition. Time spent in the social zone by NpHR- and eYFP-expressing mice across the first 5 min (inset shows total time) and social:non-social ratio following isolation. NpHR-expressing mice spent a significantly lower proportion of time in the social zone after 24 hr of isolation compared with eYFP-expressing mice (social:non-social ratio, unpaired t test: t16 = 2.236, ∗p = 0.0400, n = 7 eYFP, 11 NpHR). (D) Representative spatial heat maps showing the location of an NpHR- (upper panels) and eYFP-expressing mouse (lower panels) in the first 5 min of the task, when group-housed and following 24 hr of isolation. (E) Time spent in the social zone and social:non-social ratio of mice tested with photoinhibition while group-housed and following social isolation. Photoinhibition in NpHR-expressing mice resulted in a significant reduction in time spent in the social zone (paired t test: t10 = 2.740, ∗p = 0.0208, n = 11) and social:non-social ratio (paired t test: t10 = 2.239, p = ∗0.0491, n = 11) following social isolation, compared to when group-housed. Data are represented as mean ± SEM. See also Figure S6.CC BY · PubMed Central
Figure 7 Prior Social Experience Predicts Magnitude of Social Isolation-Induced Synaptic Potentiation and Behavioral Response to Optogenetic Manipulation (A and B) Scatter plot of AMPAR/NMDAR ratios recorded in DRN DA neurons plotted against the number of mice per cage in group-housed mice, and (B) the number of mice previously housed per cage in socially isolated mice. No significant correlation was detected in group-housed mice (Pearson’s correlation: p = 0.3653, r2 = 0.0374, n = 24), but a significant positive correlation was found in socially isolated mice (Pearson’s correlation: ∗∗p = 0.0021, r2 = 0.5282; n = 15). (C) Relative social rank of TH::Cre mice was estimated with a score of 0 indicating the most subordinate and 1 the most dominant mouse in each cage. Relative dominance of ChR2-expressing mice plotted against the change in time spent in the social zone of the three-chamber apparatus (with blue light stimulation − without stimulation). There was a significant positive correlation between relative dominance and change in social zone time (Pearson’s correlation: ∗p = 0.0163, r2 = 0.4913; n = 11). (D) Mean (+ SEM) change in social-zone time for mice of each social rank. (E) Relative dominance of ChR2-expressing mice plotted against the difference in preference for the light-stimulation zone (proportion of time spent in the stimulation zone − proportion of time spent in the unstimulated zone) in the RTPA assay. There was a significant negative correlation between relative dominance and preference for the stimulation zone (Pearson’s correlation: ∗p = 0.0338, r2 = 0.2668, n = 17). (F) Mean (− SEM) difference in preference for stimulation zone for mice of each social rank. (G) Relative dominance of NpHR-expressing mice plotted against the change in social-zone time (isolated with yellow light – grouped with yellow light). There was a significant negative correlation between relative dominance and the change in social-zone time following isolation (Pearson’s correlation: ∗∗p = 0.0038, r2 = 0.6241, n = 11). (H) Mean (± SEM) change in social zone time for mice of each social rank. See also Figure S7.
Figure 7 Prior Social Experience Predicts Magnitude of Social Isolation-Induced Synaptic Potentiation and Behavioral Response to Optogenetic Manipulation (A and B) Scatter plot of AMPAR/NMDAR ratios recorded in DRN DA neurons plotted against the number of mice per cage in group-housed mice, and (B) the number of mice previously housed per cage in socially isolated mice. No significant correlation was detected in group-housed mice (Pearson’s correlation: p = 0.3653, r2 = 0.0374, n = 24), but a significant positive correlation was found in socially isolated mice (Pearson’s correlation: ∗∗p = 0.0021, r2 = 0.5282; n = 15). (C) Relative social rank of TH::Cre mice was estimated with a score of 0 indicating the most subordinate and 1 the most dominant mouse in each cage. Relative dominance of ChR2-expressing mice plotted against the change in time spent in the social zone of the three-chamber apparatus (with blue light stimulation − without stimulation). There was a significant positive correlation between relative dominance and change in social zone time (Pearson’s correlation: ∗p = 0.0163, r2 = 0.4913; n = 11). (D) Mean (+ SEM) change in social-zone time for mice of each social rank. (E) Relative dominance of ChR2-expressing mice plotted against the difference in preference for the light-stimulation zone (proportion of time spent in the stimulation zone − proportion of time spent in the unstimulated zone) in the RTPA assay. There was a significant negative correlation between relative dominance and preference for the stimulation zone (Pearson’s correlation: ∗p = 0.0338, r2 = 0.2668, n = 17). (F) Mean (− SEM) difference in preference for stimulation zone for mice of each social rank. (G) Relative dominance of NpHR-expressing mice plotted against the change in social-zone time (isolated with yellow light – grouped with yellow light). There was a significant negative correlation between relative dominance and the change in social-zone time following isolation (Pearson’s correlation: ∗∗p = 0.0038, r2 = 0.6241, n = 11). (H) Mean (± SEM) change in social zone time for mice of each social rank. See also Figure S7.CC BY · PubMed Central
Figure S1 Electrophysiological Measures of AMPAR and NMDAR Current Properties in DRN DA Neurons of Group-Housed and Socially Isolated Mice, Related to Figure 1 (A) AMPAR/NMDAR ratios recorded in DRN DA neurons from both TH-GFP and Pitx3-GFP mice showed a significant increase following 24 hr social isolation in comparison to group-housed mice (TH-GFP – unpaired t test: t48 = 4.100, ∗∗∗p = 0.0002; n = 25 grouped, 25 isolated; Pitx3-GFP – unpaired t test: t27 = 2.813, ∗∗p = 0.0090; n = 17 grouped, 12 isolated). (B) AMPAR/NMDAR ratios recorded from GFP+/TH+ and GFP+/TH- DRN neurons, in both transgenic mouse lines, were significantly greater in socially isolated compared with group-housed mice (GFP+/TH+: unpaired t test: t46 = 4.149, ∗∗∗p = 0.0001; n = 23 grouped, 25 isolated; GFP+/TH-: unpaired t test: t8 = 2.695, ∗p = 0.0273; n = 5 grouped, 5 isolated). (C) Both adolescent (< PN50) and adult (> PN50) mice showed a significant increase in the AMPAR/NMDAR ratio following social isolation (< PN50 – unpaired t test: t40 = 3.011, ∗∗p = 0.0045; n = 23 grouped, 19 isolated; ≥ PN50 – unpaired t test: t33 = 4.226, ∗∗∗p = 0.0002; n = 18 grouped, 17 isolated). (D and E) Example high-magnification confocal images of neurobiotin-filled (blue) neurons immunohistochemically identified as TH+ (green) and (D) VIP+ (red) or (E) VIP−. Illustration of mouse housing manipulations, example AMPAR/NMDAR ratios recorded in DRN DA neurons, and bar charts showing the mean ratio for each condition. Both VIP+ and VIP− DRN DA neurons showed a significantly greater AMPAR/NMDAR ratio following social isolation (VIP+: unpaired t test: t18 = 2.210, ∗p = 0.0403; n = 10 grouped, 10 isolated; VIP−: unpaired t test: t36 = 4.78, ∗∗∗p < 0.0001; n = 18 grouped, 20 isolated). Scale bars, 20 pA, 20 ms. (F) In ACSF, the AMPAR PPR was not significantly different between naive (n = 14) and socially isolated mice (n = 11; unpaired t test: t23 = 0.0441, p = 0.952). (G) In the presence of the polyamine spermine, the AMPAR PPR was significantly greater in socially isolated relative to naive mice (unpaired t test: t31 = 2.269, ∗p = 0.0304, n = 11 naive, 22 isolated). Scale bars, 10 pA, 10 ms. (H) The decay phase of the NMDAR current was fitted with a double exponential function to calculate the weighted decay time constant (τW). No significant difference was found in the decay time constant of the NMDAR current between group-housed and socially isolated mice (Mann-Whitney U = 248, p = 0.7002, n = 28 grouped, 19 isolated). Scale bars, 20 pA, 20 ms. Data are represented as mean + SEM.
Figure S1 Electrophysiological Measures of AMPAR and NMDAR Current Properties in DRN DA Neurons of Group-Housed and Socially Isolated Mice, Related to Figure 1 (A) AMPAR/NMDAR ratios recorded in DRN DA neurons from both TH-GFP and Pitx3-GFP mice showed a significant increase following 24 hr social isolation in comparison to group-housed mice (TH-GFP – unpaired t test: t48 = 4.100, ∗∗∗p = 0.0002; n = 25 grouped, 25 isolated; Pitx3-GFP – unpaired t test: t27 = 2.813, ∗∗p = 0.0090; n = 17 grouped, 12 isolated). (B) AMPAR/NMDAR ratios recorded from GFP+/TH+ and GFP+/TH- DRN neurons, in both transgenic mouse lines, were significantly greater in socially isolated compared with group-housed mice (GFP+/TH+: unpaired t test: t46 = 4.149, ∗∗∗p = 0.0001; n = 23 grouped, 25 isolated; GFP+/TH-: unpaired t test: t8 = 2.695, ∗p = 0.0273; n = 5 grouped, 5 isolated). (C) Both adolescent (< PN50) and adult (> PN50) mice showed a significant increase in the AMPAR/NMDAR ratio following social isolation (< PN50 – unpaired t test: t40 = 3.011, ∗∗p = 0.0045; n = 23 grouped, 19 isolated; ≥ PN50 – unpaired t test: t33 = 4.226, ∗∗∗p = 0.0002; n = 18 grouped, 17 isolated). (D and E) Example high-magnification confocal images of neurobiotin-filled (blue) neurons immunohistochemically identified as TH+ (green) and (D) VIP+ (red) or (E) VIP−. Illustration of mouse housing manipulations, example AMPAR/NMDAR ratios recorded in DRN DA neurons, and bar charts showing the mean ratio for each condition. Both VIP+ and VIP− DRN DA neurons showed a significantly greater AMPAR/NMDAR ratio following social isolation (VIP+: unpaired t test: t18 = 2.210, ∗p = 0.0403; n = 10 grouped, 10 isolated; VIP−: unpaired t test: t36 = 4.78, ∗∗∗p < 0.0001; n = 18 grouped, 20 isolated). Scale bars, 20 pA, 20 ms. (F) In ACSF, the AMPAR PPR was not significantly different between naive (n = 14) and socially isolated mice (n = 11; unpaired t test: t23 = 0.0441, p = 0.952). (G) In the presence of the polyamine spermine, the AMPAR PPR was significantly greater in socially isolated relative to naive mice (unpaired t test: t31 = 2.269, ∗p = 0.0304, n = 11 naive, 22 isolated). Scale bars, 10 pA, 10 ms. (H) The decay phase of the NMDAR current was fitted with a double exponential function to calculate the weighted decay time constant (τW). No significant difference was found in the decay time constant of the NMDAR current between group-housed and socially isolated mice (Mann-Whitney U = 248, p = 0.7002, n = 28 grouped, 19 isolated). Scale bars, 20 pA, 20 ms. Data are represented as mean + SEM.CC BY · PubMed Central
Figure S2 Placement of Optic Fiber in the DRN for Photometry Recordings and Immunohistochemical Characterization of ChR2-eYFP Expression in TH::Cre and DAT::Cre Mice, Related to Figures 2 and 3 (A) The position of the tip of the optic fiber (blue cross) in the DRN of TH::Cre mice expressing GCaMP6m used for photometry recordings. (B) TH::Cre and (C) DAT::Cre mice received an injection of AAV5-DIO-ChR2-eYFP into the DRN and coronal brain sections were prepared after at least 4 weeks of expression. (D) High-magnification confocal images of eYFP+ neurons, within one anterior and one posterior section of the DRN, showing co-expression of eYFP with TH (red) but not 5-HT (cyan) in a TH::Cre and (E) a DAT::Cre mouse. White arrows indicate selected eYFP+/TH+ neurons. (F) The proportion of eYFP+ neurons in the DRN co-labeled with TH and 5-HT in TH::Cre and (G) DAT::Cre mice. There was no significant difference in the proportion of eYFP+ neurons co-labeled with TH in TH::Cre and DAT::Cre mice (Chi-square = 1.2931, p = 0.2555, n = 977/1258 neurons for TH::Cre, n = 639/801 neurons for DAT::Cre).
Figure S2 Placement of Optic Fiber in the DRN for Photometry Recordings and Immunohistochemical Characterization of ChR2-eYFP Expression in TH::Cre and DAT::Cre Mice, Related to Figures 2 and 3 (A) The position of the tip of the optic fiber (blue cross) in the DRN of TH::Cre mice expressing GCaMP6m used for photometry recordings. (B) TH::Cre and (C) DAT::Cre mice received an injection of AAV5-DIO-ChR2-eYFP into the DRN and coronal brain sections were prepared after at least 4 weeks of expression. (D) High-magnification confocal images of eYFP+ neurons, within one anterior and one posterior section of the DRN, showing co-expression of eYFP with TH (red) but not 5-HT (cyan) in a TH::Cre and (E) a DAT::Cre mouse. White arrows indicate selected eYFP+/TH+ neurons. (F) The proportion of eYFP+ neurons in the DRN co-labeled with TH and 5-HT in TH::Cre and (G) DAT::Cre mice. There was no significant difference in the proportion of eYFP+ neurons co-labeled with TH in TH::Cre and DAT::Cre mice (Chi-square = 1.2931, p = 0.2555, n = 977/1258 neurons for TH::Cre, n = 639/801 neurons for DAT::Cre).CC BY · PubMed Central
Figure S3 Placement of Optic Fiber Tip and Recording Sites for In Vivo FSCV and Evoked DA Release at Different Stimulation Frequencies, Related to Figure 3 (A) The position of the optic fiber tip (blue cross) in the DRN and (B) the recording electrode in the BNST (teal cross) and (C) CeA (magenta cross) of TH::Cre mice used for in vivo FSCV recordings. (D–E) Extracellular DA concentration ([DA]), evoked by 150 pulses of blue light (5 ms duration) at 20, 30, and 50 Hz, was quantified by calculating the area under each trace during the time window 5 s prior to stimulation onset compared to the time window 5 s post-stimulation onset. (D) Optical stimulation of DRN DA neurons evoked a significant increase in [DA] in the BNST compared to pre-stimulation levels at 20 Hz (paired t test: t44 = 5.761, ∗∗∗∗p < 0.0001), 30 Hz (paired t test: t106 = 13.74, ∗∗∗∗p < 0.0001), and 50 Hz (paired t test; t105 = 13.57, ∗∗∗∗p < 0.0001). (E) Optical stimulation of DRN DA neurons evoked a significant increase in [DA] in the CeA compared to pre-stimulation levels at 20 Hz (paired t test: t51 = 4.914, ∗∗∗∗p < 0.0001), 30 Hz (paired t test: t70 = 9.911, ∗∗∗∗p < 0.0001), and 50 Hz (paired t test: t65 = 10.64, ∗∗∗∗p < 0.0001). (F) As a group, peak [DA] was increased in a frequency-dependent manner (two-way ANOVA: F2,441 = 22.19, ∗∗∗∗p < 0.0001). In the BNST, peak [DA] evoked by 30 Hz and 50 Hz stimulation was greater than that evoked by 20 Hz (Bonferroni multiple comparisons: 20 Hz versus 30 Hz, ∗∗∗∗p < 0.0001; 20 Hz versus 50 Hz, ∗∗∗∗p < 0.0001; 30 Hz versus 50 Hz, p > 0.9999). In contrast, peak [DA] in the CeA did not differ significantly for different stimulation frequencies (Bonferroni multiple comparisons: 20 Hz versus 30 Hz, p = 0.7134; 20 Hz versus 50 Hz, p = 0.0806; 30 Hz versus 50 Hz, p > 0.9999). Peak-evoked [DA] was greater in the BNST compared to the CeA at 30 Hz (F1,441 = 54.39, ∗∗∗∗p < 0.0001; Bonferroni multiple comparisons: ∗∗∗∗p < 0.0001) and 50 Hz (Bonferroni multiple comparisons: ∗∗∗∗p < 0.0001). Data are represented as mean + SEM.
Figure S3 Placement of Optic Fiber Tip and Recording Sites for In Vivo FSCV and Evoked DA Release at Different Stimulation Frequencies, Related to Figure 3 (A) The position of the optic fiber tip (blue cross) in the DRN and (B) the recording electrode in the BNST (teal cross) and (C) CeA (magenta cross) of TH::Cre mice used for in vivo FSCV recordings. (D–E) Extracellular DA concentration ([DA]), evoked by 150 pulses of blue light (5 ms duration) at 20, 30, and 50 Hz, was quantified by calculating the area under each trace during the time window 5 s prior to stimulation onset compared to the time window 5 s post-stimulation onset. (D) Optical stimulation of DRN DA neurons evoked a significant increase in [DA] in the BNST compared to pre-stimulation levels at 20 Hz (paired t test: t44 = 5.761, ∗∗∗∗p < 0.0001), 30 Hz (paired t test: t106 = 13.74, ∗∗∗∗p < 0.0001), and 50 Hz (paired t test; t105 = 13.57, ∗∗∗∗p < 0.0001). (E) Optical stimulation of DRN DA neurons evoked a significant increase in [DA] in the CeA compared to pre-stimulation levels at 20 Hz (paired t test: t51 = 4.914, ∗∗∗∗p < 0.0001), 30 Hz (paired t test: t70 = 9.911, ∗∗∗∗p < 0.0001), and 50 Hz (paired t test: t65 = 10.64, ∗∗∗∗p < 0.0001). (F) As a group, peak [DA] was increased in a frequency-dependent manner (two-way ANOVA: F2,441 = 22.19, ∗∗∗∗p < 0.0001). In the BNST, peak [DA] evoked by 30 Hz and 50 Hz stimulation was greater than that evoked by 20 Hz (Bonferroni multiple comparisons: 20 Hz versus 30 Hz, ∗∗∗∗p < 0.0001; 20 Hz versus 50 Hz, ∗∗∗∗p < 0.0001; 30 Hz versus 50 Hz, p > 0.9999). In contrast, peak [DA] in the CeA did not differ significantly for different stimulation frequencies (Bonferroni multiple comparisons: 20 Hz versus 30 Hz, p = 0.7134; 20 Hz versus 50 Hz, p = 0.0806; 30 Hz versus 50 Hz, p > 0.9999). Peak-evoked [DA] was greater in the BNST compared to the CeA at 30 Hz (F1,441 = 54.39, ∗∗∗∗p < 0.0001; Bonferroni multiple comparisons: ∗∗∗∗p < 0.0001) and 50 Hz (Bonferroni multiple comparisons: ∗∗∗∗p < 0.0001). Data are represented as mean + SEM.CC BY · PubMed Central
Figure S4 Optically Evoked Glutamate Currents in the BNST and CeA Following DRN DA Terminal Stimulation in TH::Cre and DAT::Cre Mice, Related to Figure 4 (A and B) Location of ex vivo recorded cells in the BNST and (C and D) CeA of TH::Cre and DAT::Cre mice, superimposed on coronal brain atlas sections (distance from Bregma shown in top left corner). The relative amplitude of the optically evoked EPSC from each cell is indicated by the size of the teal (BNST) or magenta (CeA) circle. Cells with no response are displayed in gray. (E) Pie charts showing the proportion of cells that responded with an EPSC to a 5 ms blue light pulse in the BNST and CeA of TH::Cre and DAT::Cre mice and (F) the amplitude and (G) latency of each EPSC. There was no significant difference in the proportion of neurons which responded to optical stimulation with an EPSC in TH::Cre mice compared to DAT::Cre mice in the BNST (Chi-square = 0.0271, p = 0.8691) or CeA (Chi-square = 0.1544, p = 0.6944). Data are represented as mean + SEM
Figure S4 Optically Evoked Glutamate Currents in the BNST and CeA Following DRN DA Terminal Stimulation in TH::Cre and DAT::Cre Mice, Related to Figure 4 (A and B) Location of ex vivo recorded cells in the BNST and (C and D) CeA of TH::Cre and DAT::Cre mice, superimposed on coronal brain atlas sections (distance from Bregma shown in top left corner). The relative amplitude of the optically evoked EPSC from each cell is indicated by the size of the teal (BNST) or magenta (CeA) circle. Cells with no response are displayed in gray. (E) Pie charts showing the proportion of cells that responded with an EPSC to a 5 ms blue light pulse in the BNST and CeA of TH::Cre and DAT::Cre mice and (F) the amplitude and (G) latency of each EPSC. There was no significant difference in the proportion of neurons which responded to optical stimulation with an EPSC in TH::Cre mice compared to DAT::Cre mice in the BNST (Chi-square = 0.0271, p = 0.8691) or CeA (Chi-square = 0.1544, p = 0.6944). Data are represented as mean + SEMCC BY · PubMed Central
Figure S5 Effect of Photoactivation in TH::Cre and DAT::Cre Mice on Behavioral Measures of Social Preference, Avoidance, Arousal, and Anxiety-Related Behavior, Related to Figures 5 and 7 (A) The position of the optic fiber tip above the DRN of TH::Cre and DAT::Cre mice expressing ChR2 or eYFP used for behavioral analysis. (B) Example confocal images showing the optic fiber track (white dashed line), ChR2-eYFP expression (green), and post hoc immunohistochemistry for TH (red) in the DRN of TH::Cre mice at each anteroposterior position indicated in A. (C) Time spent in the social zone of the three-chamber apparatus and (D) social:non-social ratio of individual TH::Cre and DAT::Cre mice expressing ChR2 (green and blue, respectively) or eYFP (gray and brown, respectively) in DRN DA neurons. There was no significant difference in the behavior of TH::Cre and DAT::Cre mice with blue light stimulation (ChR2: time in social zone – unpaired t test: t17 = 0.0565, p = 0.9556; social:non-social ratio – t17 = 1.185, p = 0.2523, n = 11 TH::Cre, 8 DAT::Cre; eYFP: time in social zone – unpaired t test: t15 = 0.0165, p = 0.9870; social:non-social ratio – t15 = 0.3015, p = 0.7672, n = 11 TH::Cre, 6 DAT::Cre) or without stimulation (ChR2: time in social zone – unpaired t test: t17 = 0.1162, p = 0.9089; social:non-social ratio – t17 = 0.7161, p = 0.4836, n = 11 TH::Cre, 8 DAT::Cre; eYFP: time in social zone – unpaired t test: t15 = 0.8873, p = 0.3889; social:non-social ratio – t15 = 0.8239, p = 0.4229, n = 11 TH::Cre, 6 DAT::Cre). Pooling data from TH::Cre and DAT::Cre mice revealed that blue light stimulation caused a significant increase in the time spent in the social zone and social:non-social ratio of ChR2- (time in social zone – paired t test: t18 = 3.877, ∗∗p = 0.0011; social:non-social ratio – t18 = 4.527, ∗∗∗p = 0.0003, n = 19), but not eYFP-expressing mice (time in social zone – paired t test: t16 = 0.4094, p = 0.6876; social:non-social ratio – t16 = 0.5364, p = 0.5991, n = 17). (E) In an ICSS task, there was no significant difference in the number of nose pokes made into the active or inactive ports by TH::Cre and DAT::Cre mice expressing ChR2 (active – unpaired t test: t10 = 0.2347, p = 0.8192; inactive – unpaired t test: t10 = 0.3284, p = 0.7494, n = 5 TH::Cre, 7 DAT::Cre) or eYFP (active – unpaired t test: t19 = 0.2205, p = 0.8304; inactive – unpaired t test: t9 = 0.7528, p = 0.4708, n = 7 TH::Cre, 4 DAT::Cre). (F) The proportion of time spent in the stimulation zone during an RTPA assay was not significantly difference between TH::Cre and DAT::Cre mice expressing ChR2- (unpaired t test: t26 = 0.1678, p = 0.8681, n = 17 TH::Cre, 11 DAT::Cre) or eYFP (unpaired t test: t15 = 0.5274, p = 0.6056, n = 11 TH::Cre, 6 DAT::Cre). Pooling data from TH::Cre and DAT::Cre mice revealed that ChR2- but not eYFP-expressing mice spent a significantly greater proportion of time in the unstimulated zone (ChR2 – paired t test: t27 = 4.033, ∗∗∗p = 0.0004, n = 28; eYFP: paired t test: t16 = 0.5849, p = 0.5668, n = 17). (G) Representative track from a ChR2-expressing and (H) eYFP-expressing TH::Cre mouse in the RTPA assay. ChR2- (paired t test: t16 = 3.248, ∗∗p = 0.005, n = 17), but not eYFP-expressing TH::Cre mice (paired t test: t10 = 0.6779, p = 0.5132, n = 11) spent a significantly lower proportion of time in the light-paired zone of the chamber. (I and J) There was no effect of blue light stimulation on locomotion in the open field as measured by total distance traveled in the arena (TH::Cre – two-way ANOVA: group × light interaction: F2,34 = 0.6333, p = 0.5370, n = 10 ChR2, 9 eYFP; DAT::Cre – two-way ANOVA: group × light interaction: F2,30 = 0.2483, p = 0.7817, n = 10 ChR2, 7 eYFP). (K-N) Novelty preference was assessed in a modified three-chamber task in which a novel object was placed under one inverted cup instead of a juvenile mouse. Optical stimulation of DRN DA neurons did not significantly affect time spent exploring the novelty zone in (K and L) ChR2- (TH::Cre – paired t test: t4 = 0.9499, p = 0.3959, n = 5; DAT::Cre – paired t test: t8 = 0.1215, p = 0.9063, n = 9) or (M and N) eYFP-expressing mice (TH::Cre – paired t test: t5 = 0.8107, p = 0.4544, n = 6; DAT::Cre – paired t test: t5 = 0.6382, p = 0.5514, n = 6). (O) Mice were assessed for anxiety-related behavior in the elevated plus maze (EPM) and (P) open field test across a 15 min trial with the middle 5 min paired with blue light stimulation. Representative tracks from a ChR2- (green) and an eYFP-expressing (gray) TH::Cre mouse showing the first ‘light OFF’ and second ‘light ON’ epochs. (Q–T) There was no significant effect of light stimulation on (Q and S) the time spent in the open arms of the EPM in ChR2- relative to eYFP-expressing mice (TH::Cre - two-way ANOVA: group × light interaction: F2,34 = 3.016, p = 0.0623, n = 10 ChR2, 9 eYFP; DAT::Cre – two-way ANOVA: group × light interaction: F2,32 = 0.3468, p = 0.7095, n = 11 ChR2, 7 eYFP) or (R and T) the time spent in the center of the open field (TH::Cre – two-way ANOVA: group × light interaction: F2,34 = 0.1201, p = 0.8872, n = 10 ChR2, 9 eYFP; DAT::Cre – two-way ANOVA: group × light interaction: F2,30 = 0.2322, p = 0.7942, n = 10 ChR2, 7 eYFP). (U and V) In a non-exploratory assay for anxiety-related behavior, the resident-intruder task, blue light stimulation had no effect on total time spent sniffing the juvenile intruder mouse (TH::Cre – ChR2: paired t test: t11 = 0.1924, p = 0.8510, n = 12; eYFP: t11 = 0.2378, p = 0.8164; n = 12; DAT::Cre – ChR2: paired t test: t6 = 0.01584, p = 0.9879, n = 7; eYFP: t4 = 1.158, p = 0.3113; n = 5) or (W and X) rearing (TH::Cre – ChR2: t11 = 0.1012, p = 0.9213; n = 12; eYFP: t11 = 0.1133, p = 0.9118, n = 12; DAT::Cre – ChR2: paired t test: t6 = 0.02656, p = 0.9797, n = 7; eYFP: t4 = 0.9856, p = 0.3801; n = 5). Data are represented as mean ± SEM.
Figure S5 Effect of Photoactivation in TH::Cre and DAT::Cre Mice on Behavioral Measures of Social Preference, Avoidance, Arousal, and Anxiety-Related Behavior, Related to Figures 5 and 7 (A) The position of the optic fiber tip above the DRN of TH::Cre and DAT::Cre mice expressing ChR2 or eYFP used for behavioral analysis. (B) Example confocal images showing the optic fiber track (white dashed line), ChR2-eYFP expression (green), and post hoc immunohistochemistry for TH (red) in the DRN of TH::Cre mice at each anteroposterior position indicated in A. (C) Time spent in the social zone of the three-chamber apparatus and (D) social:non-social ratio of individual TH::Cre and DAT::Cre mice expressing ChR2 (green and blue, respectively) or eYFP (gray and brown, respectively) in DRN DA neurons. There was no significant difference in the behavior of TH::Cre and DAT::Cre mice with blue light stimulation (ChR2: time in social zone – unpaired t test: t17 = 0.0565, p = 0.9556; social:non-social ratio – t17 = 1.185, p = 0.2523, n = 11 TH::Cre, 8 DAT::Cre; eYFP: time in social zone – unpaired t test: t15 = 0.0165, p = 0.9870; social:non-social ratio – t15 = 0.3015, p = 0.7672, n = 11 TH::Cre, 6 DAT::Cre) or without stimulation (ChR2: time in social zone – unpaired t test: t17 = 0.1162, p = 0.9089; social:non-social ratio – t17 = 0.7161, p = 0.4836, n = 11 TH::Cre, 8 DAT::Cre; eYFP: time in social zone – unpaired t test: t15 = 0.8873, p = 0.3889; social:non-social ratio – t15 = 0.8239, p = 0.4229, n = 11 TH::Cre, 6 DAT::Cre). Pooling data from TH::Cre and DAT::Cre mice revealed that blue light stimulation caused a significant increase in the time spent in the social zone and social:non-social ratio of ChR2- (time in social zone – paired t test: t18 = 3.877, ∗∗p = 0.0011; social:non-social ratio – t18 = 4.527, ∗∗∗p = 0.0003, n = 19), but not eYFP-expressing mice (time in social zone – paired t test: t16 = 0.4094, p = 0.6876; social:non-social ratio – t16 = 0.5364, p = 0.5991, n = 17). (E) In an ICSS task, there was no significant difference in the number of nose pokes made into the active or inactive ports by TH::Cre and DAT::Cre mice expressing ChR2 (active – unpaired t test: t10 = 0.2347, p = 0.8192; inactive – unpaired t test: t10 = 0.3284, p = 0.7494, n = 5 TH::Cre, 7 DAT::Cre) or eYFP (active – unpaired t test: t19 = 0.2205, p = 0.8304; inactive – unpaired t test: t9 = 0.7528, p = 0.4708, n = 7 TH::Cre, 4 DAT::Cre). (F) The proportion of time spent in the stimulation zone during an RTPA assay was not significantly difference between TH::Cre and DAT::Cre mice expressing ChR2- (unpaired t test: t26 = 0.1678, p = 0.8681, n = 17 TH::Cre, 11 DAT::Cre) or eYFP (unpaired t test: t15 = 0.5274, p = 0.6056, n = 11 TH::Cre, 6 DAT::Cre). Pooling data from TH::Cre and DAT::Cre mice revealed that ChR2- but not eYFP-expressing mice spent a significantly greater proportion of time in the unstimulated zone (ChR2 – paired t test: t27 = 4.033, ∗∗∗p = 0.0004, n = 28; eYFP: paired t test: t16 = 0.5849, p = 0.5668, n = 17). (G) Representative track from a ChR2-expressing and (H) eYFP-expressing TH::Cre mouse in the RTPA assay. ChR2- (paired t test: t16 = 3.248, ∗∗p = 0.005, n = 17), but not eYFP-expressing TH::Cre mice (paired t test: t10 = 0.6779, p = 0.5132, n = 11) spent a significantly lower proportion of time in the light-paired zone of the chamber. (I and J) There was no effect of blue light stimulation on locomotion in the open field as measured by total distance traveled in the arena (TH::Cre – two-way ANOVA: group × light interaction: F2,34 = 0.6333, p = 0.5370, n = 10 ChR2, 9 eYFP; DAT::Cre – two-way ANOVA: group × light interaction: F2,30 = 0.2483, p = 0.7817, n = 10 ChR2, 7 eYFP). (K-N) Novelty preference was assessed in a modified three-chamber task in which a novel object was placed under one inverted cup instead of a juvenile mouse. Optical stimulation of DRN DA neurons did not significantly affect time spent exploring the novelty zone in (K and L) ChR2- (TH::Cre – paired t test: t4 = 0.9499, p = 0.3959, n = 5; DAT::Cre – paired t test: t8 = 0.1215, p = 0.9063, n = 9) or (M and N) eYFP-expressing mice (TH::Cre – paired t test: t5 = 0.8107, p = 0.4544, n = 6; DAT::Cre – paired t test: t5 = 0.6382, p = 0.5514, n = 6). (O) Mice were assessed for anxiety-related behavior in the elevated plus maze (EPM) and (P) open field test across a 15 min trial with the middle 5 min paired with blue light stimulation. Representative tracks from a ChR2- (green) and an eYFP-expressing (gray) TH::Cre mouse showing the first ‘light OFF’ and second ‘light ON’ epochs. (Q–T) There was no significant effect of light stimulation on (Q and S) the time spent in the open arms of the EPM in ChR2- relative to eYFP-expressing mice (TH::Cre - two-way ANOVA: group × light interaction: F2,34 = 3.016, p = 0.0623, n = 10 ChR2, 9 eYFP; DAT::Cre – two-way ANOVA: group × light interaction: F2,32 = 0.3468, p = 0.7095, n = 11 ChR2, 7 eYFP) or (R and T) the time spent in the center of the open field (TH::Cre – two-way ANOVA: group × light interaction: F2,34 = 0.1201, p = 0.8872, n = 10 ChR2, 9 eYFP; DAT::Cre – two-way ANOVA: group × light interaction: F2,30 = 0.2322, p = 0.7942, n = 10 ChR2, 7 eYFP). (U and V) In a non-exploratory assay for anxiety-related behavior, the resident-intruder task, blue light stimulation had no effect on total time spent sniffing the juvenile intruder mouse (TH::Cre – ChR2: paired t test: t11 = 0.1924, p = 0.8510, n = 12; eYFP: t11 = 0.2378, p = 0.8164; n = 12; DAT::Cre – ChR2: paired t test: t6 = 0.01584, p = 0.9879, n = 7; eYFP: t4 = 1.158, p = 0.3113; n = 5) or (W and X) rearing (TH::Cre – ChR2: t11 = 0.1012, p = 0.9213; n = 12; eYFP: t11 = 0.1133, p = 0.9118, n = 12; DAT::Cre – ChR2: paired t test: t6 = 0.02656, p = 0.9797, n = 7; eYFP: t4 = 0.9856, p = 0.3801; n = 5). Data are represented as mean ± SEM.CC BY · PubMed Central
Figure S6 Effect of Photoinhibition in TH::Cre Mice on Behavioral Measures of Arousal and Anxiety-Related Behavior, Related to Figure 6 (A) The position of the optic fiber tip above the DRN of TH::Cre mice expressing NpHR (orange crosses) or eYFP (gray crosses) used for behavioral analysis. (B) No significant difference was detected between NpHR- and eYFP-expressing mice in distance traveled in the open field. (C) In an assay for novelty preference, optical inhibition of DRN DA neurons had no effect on the time spent in the novelty zone in NpHR- (paired t test: t5 = 0.08958, p = 0.9321, n = 6) or (D) eYFP-expressing mice (paired t test: t6 = 0.4595, p = 0.6620; n = 7). (E) Representative track from a TH::Cre mouse expressing NpHR in DRN DA neurons in the EPM and (F) open field before light stimulation and during delivery of constant yellow light. (G) There was no significant effect of photoinhibition on time spent in the open arms of the EPM (two-way ANOVA: group × light interaction: F2,38 = 1.009, p = 0.3742, n = 11 NpHR, 10 eYFP) or (H) time spent in the center of the open field (two-way ANOVA: group × light interaction: F2,46 = 2.138, p = 0.1294, n = 13 NpHR, 12 eYFP) in NpHR- relative to eYFP-expressing mice. (I) In the resident-intruder task optical inhibition did not significantly affect time spent sniffing the juvenile (NpHR: paired t test – t13 = 0.8025, p = 0.4367, n = 14; eYFP: t9 = 1.628, p = 0.1380; n = 10) or (J) time spent rearing (NpHR: t13 = 0.0619, p = 0.9516, n = 14; eYFP: t9 = 1.679, p = 0.1274; n = 10). Data are represented as mean ± SEM.
Figure S6 Effect of Photoinhibition in TH::Cre Mice on Behavioral Measures of Arousal and Anxiety-Related Behavior, Related to Figure 6 (A) The position of the optic fiber tip above the DRN of TH::Cre mice expressing NpHR (orange crosses) or eYFP (gray crosses) used for behavioral analysis. (B) No significant difference was detected between NpHR- and eYFP-expressing mice in distance traveled in the open field. (C) In an assay for novelty preference, optical inhibition of DRN DA neurons had no effect on the time spent in the novelty zone in NpHR- (paired t test: t5 = 0.08958, p = 0.9321, n = 6) or (D) eYFP-expressing mice (paired t test: t6 = 0.4595, p = 0.6620; n = 7). (E) Representative track from a TH::Cre mouse expressing NpHR in DRN DA neurons in the EPM and (F) open field before light stimulation and during delivery of constant yellow light. (G) There was no significant effect of photoinhibition on time spent in the open arms of the EPM (two-way ANOVA: group × light interaction: F2,38 = 1.009, p = 0.3742, n = 11 NpHR, 10 eYFP) or (H) time spent in the center of the open field (two-way ANOVA: group × light interaction: F2,46 = 2.138, p = 0.1294, n = 13 NpHR, 12 eYFP) in NpHR- relative to eYFP-expressing mice. (I) In the resident-intruder task optical inhibition did not significantly affect time spent sniffing the juvenile (NpHR: paired t test – t13 = 0.8025, p = 0.4367, n = 14; eYFP: t9 = 1.628, p = 0.1380; n = 10) or (J) time spent rearing (NpHR: t13 = 0.0619, p = 0.9516, n = 14; eYFP: t9 = 1.679, p = 0.1274; n = 10). Data are represented as mean ± SEM.CC BY · PubMed Central
Figure S7 Social Rank Predicts the Behavioral Effect of Photoactivation and Photoinhibition of DRN DA Neurons, Related to Figure 7 (A) Proportion of ‘wins’ in the tube test for social dominance plotted against the relative body weight of TH::Cre mice. There was a significant positive correlation between the proportion of wins and body weight (Spearman’s rank correlation: ∗∗∗p = 0.0008, r2 = 0.1869; n = 57). (B) Relative body weight of all mice classified as subordinate, intermediate, and dominant based on performance in the tube test. (C) Time spent by ChR2-expressing mice in the social zone of the three-chamber apparatus, with and without blue light stimulation, ranked based on relative body weight. (D) % time spent in the light-paired and unstimulated zones in the RTPA assay, by ChR2-expressing mice ranked based on relative body weight. (E) Time spent by NpHR-expressing mice in the social zone of the three-chamber apparatus when receiving yellow light while group-housed and following social isolation, ranked based on relative weight or (F) proportion of wins in the tube test. Data are represented as mean +SEM. Paired t tests used for within-group comparisons, ∗p < 0.05, ∗∗p < 0.01.
Figure S7 Social Rank Predicts the Behavioral Effect of Photoactivation and Photoinhibition of DRN DA Neurons, Related to Figure 7 (A) Proportion of ‘wins’ in the tube test for social dominance plotted against the relative body weight of TH::Cre mice. There was a significant positive correlation between the proportion of wins and body weight (Spearman’s rank correlation: ∗∗∗p = 0.0008, r2 = 0.1869; n = 57). (B) Relative body weight of all mice classified as subordinate, intermediate, and dominant based on performance in the tube test. (C) Time spent by ChR2-expressing mice in the social zone of the three-chamber apparatus, with and without blue light stimulation, ranked based on relative body weight. (D) % time spent in the light-paired and unstimulated zones in the RTPA assay, by ChR2-expressing mice ranked based on relative body weight. (E) Time spent by NpHR-expressing mice in the social zone of the three-chamber apparatus when receiving yellow light while group-housed and following social isolation, ranked based on relative weight or (F) proportion of wins in the tube test. Data are represented as mean +SEM. Paired t tests used for within-group comparisons, ∗p < 0.05, ∗∗p < 0.01.CC BY · PubMed Central

Verification

Claims the paper makes

54 Own result · 37 Not checked yet · 26 Own interpretation · 1 Own method · 1 No basis given

Verify all 62 references — free account

Show all 87 claims

  • It may state two separate claims as one. 13
  • Probably not a substantial claim. 20
  • The quote only partly states this claim. 1
32 of 32

  • The paper relies on this without support.

    The behavioral and neural state induced by acute social isolation in mice corresponds to the subjective experience of loneliness in humans.

    “Taken together, we propose that DRN DA neurons represent a neural substrate for the subjective experience of social isolation and serve to promote a response to alleviate this aversive state.”
    “supporting a role for DRN dopamine neurons in mediating a loneliness-like state.”

  • The paper relies on this without support.

    The subjective experience of social interaction differs between members of a social group depending on their social rank.

    “As a result, the subjective experience of social interaction is likely to differ between members of a social group dependent on their social rank.”

  • The paper relies on this without support.

    Social rank offers a useful estimate of an individual's subjective social experience.

    “social rank offers a useful estimate of an individual’s subjective social experience.”
3 of 3

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

Citations

Cites

Show all 73 references

Cited by

Sorted by citations

Related

See all

Metadata

Authors
Matthews, G. A., Nieh, E. H., Vander Weele, C. M., Halbert, S. A., Pradhan, R. V., Yosafat, A. S., Glober, G. F., Izadmehr, E. M., Thomas, R. E., Lacy, G. D., Wildes, C. P., Ungless, M. A., & Tye, K. M.
Affiliations
  • Hammersmith Hospital (GB)
  • Massachusetts Institute of Technology (US)
Corresponding author
  • Ungless, Mark A.
  • Tye, Kay M.
ORCID
Venue
Cell
Publisher
Elsevier BV
Published
2016-02
Volume
164
Issue
4
Pages
617-631
ISSN
0092-8674 (ISSN-L)
DOI
10.1016/j.cell.2015.12.040
PMC
PMC4752823
OpenAlex
W2269038554
Semantic Scholar
1adbaade5ef3bde98d2bb90790ddf3f098fbafb7
Cited by
402
References
73
Citations by year
2026: 30 · 2025: 47 · 2024: 46 · 2023: 48 · 2022: 36 · 2021: 56 · 2020: 30 · 2019: 34 · 2018: 33 · 2017: 25 · 2016: 17
FWCI
20.38
Type
journal article
Language
en
Open access
Licence
Locations
  • Cell (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DSpace@MIT (Massachusetts Institute of Technology) (submittedVersion, cc-by-nc)
  • Spiral (Imperial College London) (submittedVersion, other-oa)
  • Elsevier eBooks
Funders
  • JPB Foundation
  • PIIF
  • PNDRF
  • JFDP
  • Whitehall Foundation
  • Klingenstein Foundation
  • NARSAD Young Investigator Award
  • Alfred P Sloan Foundation
  • New York Stem Cell Foundation
  • McKnight Foundation
  • Whitehead Career Development Chair
  • NIMH (R01-MH102441-01)
  • NIA (RF1-AG047661-01)
  • NIH (DP2-DK-102256-01)
  • U.K. Medical Research Council (MC-A654-5QB70)
  • The Royal Society
  • Simons Center
  • NSF Graduate Research Program Fellowship
  • Integrative Neuronal Systems Fellowship
  • NIGMS (T32 GM007484)
  • NSF
  • Undergraduate Research Opportunities Program at MIT
  • MIT Summer Research Program
  • Northeastern Cooperative Education Program
Topics
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neurotransmitter Receptor Influence on Behavior
  • Life Sciences › Neuroscience › Endocrine and Autonomic Systems › Neuroendocrine regulation and behavior
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neuroscience and Neuropharmacology Research
Keywords
dorsal raphe nucleus, dopaminergic neurons, social isolation, loneliness, social reward, social preferences, place avoidance, social rank, synaptic plasticity, calcium imaging, optogenetic stimulation, negative emotions
MeSH terms
Animals, Dopamine, Loneliness, Male, Reward, Synapses, Ventral Tegmental Area, Patch-Clamp Techniques, Glutamic Acid, Mice, Dopaminergic Neurons, Optogenetics, Dorsal Raphe Nucleus, In Vitro Techniques

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

A language model drew the claims from the paper on 2026-10-09, under version 3 of the claim definition (model deepseek-v4.1-flash, prompt extract-v3).

A second model labelled each claim on 2026-10-09 (model jev-1.13-20260917, definition revision gold-guideline-v0.2).

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review