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A neural circuit for comorbid depressive symptoms in chronic pain

A Publisher Correction to this article was published on 01 October 2019

This article has been updated

Abstract

Comorbid depressive symptoms (CDS) in chronic pain are a common health problem, but the neural circuit mechanisms underlying these symptoms remain unclear. Here we identify a novel pathway involving 5-hydroxytryptamine (5-HT) projections from the dorsal raphe nucleus (5-HTDRN) to somatostatin (SOM)-expressing and non-SOM interneurons in the central nucleus of the amygdala (CeA). The SOMCeA neurons project directly to the lateral habenula, an area known involved in depression. Inhibition of the 5-HTDRN→SOMCeA pathway produced depression-like behavior in a male mouse model of chronic pain. Activation of this pathway using pharmacological or optogenetic approaches reduced depression-like behavior in these mice. Human functional magnetic resonance imaging data showed that compared to healthy controls, functional connectivity between the CeA-containing centromedial amygdala and the DRN was reduced in patients with CDS but not in patients in chronic pain without depression. These findings indicate that a novel 5-HTDRN→SOMCeA→lateral habenula pathway may mediate at least some aspects of CDS.

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Fig. 1: Dissection of the 5-HTDRN→SOMCeA circuit.
Fig. 2: 5-HTDRN→Non-SOMCeA circuits.
Fig. 3: Decreased 5-HTDRN inputs to the SOMCeA circuit in mice with chronic pain.
Fig. 4: Activation of the 5-HTDRN→SOMCeA circuit reduces depressive-like behavior in mice with chronic pain.
Fig. 5: Increased SOMCeA inputs to the GluLHb circuit in in mice with chronic pain.
Fig. 6: Dissection of the 5-HTDRN→SOMCeA→GluLHb circuit.

Data availability

The data that support the findings of this study are available from the corresponding author upon request.

Change history

  • 01 October 2019

    An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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Acknowledgements

We thank X. Yu and Y. Yang for providing Ai9 (RCL-tdT) mice. We thank F. Xu and T. Xue for technical support. All data are available in the main text or the Supplementary Data. Support for this study was provided by the National Natural Science Foundation of China (grant nos. 81870877, 91732303 and 91849119 to Z.Z.); the National Key Research and Development Program of China (grant no. 2016YFC1305900 to Z.Z.); the Strategic Priority Research Program of the Chinese Academy of Sciences (grant no. XDB02010000 to Z.Z.) and the National Natural Science Foundation of China (grant no. 31600851 to W.T. and grant no. 81600964 to Y.J.).

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Authors

Contributions

W.Z., Y.J., Q.M. and X.Zhu designed the studies, performed most of the experiments and data analysis, and wrote the draft manuscript. T.B., Y.T., Y.M., L.W., H.Z., B.-S.Q., X.Zhang, Y.L. and W.X. conducted the fMRI experiments and data analyses and wrote the text of the final manuscript. W.T., H.W., Jie Li and N.Z. conducted some of the molecular and behavioral experiments. X.L., M.-H.L., Juan Li and H.X. designed the viral tracing experiments in CR-Cre, PV-Ce and SOM-Cre mice. Patients with chronic pain or depression were evaluated by certified doctors Y.M., Y.T., W.X. and K.W. J.Z., K.W., G.R.L., L.X. and Z.Z. were involved in the overall design of the study and the revision of the final manuscript. Z.Z. was involved in the overall design of the project, individual experiments, data analysis and the writing of the final manuscript.

Corresponding author

Correspondence to Zhi Zhang.

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The authors declare no competing interests.

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Peer review information: Nature Neuroscience thanks Rohini Kuner, Manuel Mameli and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Integrated supplementary information

Supplementary Figure 1 Mapping of CeA GABA neuron inputs.

a, Schematic of viral injection in the CeA of GAD2-Cre mice. b, Representative images of viral expression within the CeA (left) and DsRed signals in the BLA and paraventricular thalamus (PVT) (right). Scale bars: 500 µm (left), 50 µm (right). c,d, DsRed signals in the posterior thalamus (Po), parafascicular thalamic nucleus (PF, c), and DRN (d). Scale bars: 300 µm (left), 50 µm (right). Aq, aqueduct. e,f, CeA injection of Cre-dependent helper virus (AAV-DIO-TVA-GFP, AAV-DIO-RVG, green) and RV-EnvA-ΔG-DsRed (red) in CR-Cre mice (e) or PV-Cre mice (f). No DsRed signals were identified in the DRN. Scale bars: 200 µm (left), 50 µm (right). CR, calretinin; PV, parvalbumin. g, Observed mCherry fibers projected from 5-HTDRN neurons in the CeA. Scale bar: 200 µm. h, Sample traces of action potentials evoked by 473 nm light (blue bars) recorded from DRN mCherry+ neurons in acute slices. i, Representative images of DRN infusion of AAV-DIO-ChR2-mCherry in Pet1-Cre mice, and mCherry+ neurons in the DRN that co-localized with the 5-HT neuronal marker TPH2. Scale bar: 200 µm. j, Statistical data of mCherry+ neurons in the DRN that co-localized with TPH2 (n = 9 slices from three mice). Data are shown as box and whisker plots [medians, quartiles (boxes) and ranges min to max (whiskers)]. For b-i, the experiment was independently repeated five times with similar results obtained.

Supplementary Figure 2 Effects of the GABAAR blocker picrotoxin on 5-HTDRN→SOMCeA synaptic transmission.

a, Schematic of the DRN injection of AAV-hSyn-ChR2-EYFP in SOM-tdTOM mice and the recording configuration in acute slices. b, A representative image of viral expression within the DRN. The experiment was independently repeated five times with similar results obtained. Scale bar, 250 µm. c, A sample trace and summarized data of hyperpolarized potentials of SOMCeA neurons evoked by photostimulation in the presence of picrotoxin (n = 5 cells, t4 = 0.03337, P = 0.975). Significance was assessed by two-tailed paired Student’s t test in c. The data are expressed as the mean ± s.e.m. ns, no significance.

Supplementary Figure 3 SOMCeA neurons are innervated by 5-HTDRN neurons.

a,b, Representative images of c-Fos expression in the CeA after optical inhibition of 5-HTDRN→SOMCeA circuit of mice with the DRN infusion of eNpHR3.0-EYFP (a) or EYFP (b). The experiment was independently repeated four times with similar results obtained. Scale bars, 500 μm (left), 50 μm (right). c, Summarized data of c-Fos-positive neurons in the CeA from the indicated groups (n = 9 slices from four mice each group; t16 = 9.119, P < 0.0001). d,e, Representative images (d) and summarized data (e) of c-Fos-positive neurons that co-localized with SOM immunofluorescence in the CeA (n = 9 slices from three mice). Scale bar, 50 μm. f,g, Sample traces (f) and summarized data (g) of the spontaneous action potential firing rate showing the effect of optical activation of 5-HTDRN terminals in the CeA with or without perfusion of WAY (n = 10 cells each group; F1,18 = 10.69, P = 0.0043). h, Representative images and percentage of 5-HT2AR immunoreactivity in non-SOM neurons in the CeA (n = 9 slice from three mice). Scale bar, 50 μm. For e and h, data are shown as box and whisker plots [medians, quartiles (boxes) and ranges min to max (whiskers)]. Significance was assessed by two-tailed unpaired Student’s t test in c, and two-way RM ANOVA with post hoc comparison between groups in g. The data are expressed as the mean ± s.e.m. ***P < 0.001. ns, no significance.

Supplementary Figure 4 Behavioural phenotypes in mice treated with CFA or SNI.

a, Time course of CFA-induced sensory pain (saline, n = 8 mice; CFA, n = 13 mice; F8,169 = 4.181, P = 0.0001). b, Performance of sham 2W and SNI 2W mice in SPT, TST, OFT, and EPM (sham 2W, n = 10 mice; SNI 2W, n = 15 mice; SPT, t23 = 0.0424, P = 0.9666; TST, t23 = 0.490, P = 0.6291; OFT, t23 = 1.388, P = 0.1783; EPM, t23 = 0.4599, P = 0.6499). c, Performance of mice treated with CFA or saline for two or three weeks in SPT, TST, OFT, and EPM (n = 12 mice per group; SPT, F3,44 = 4.261, P = 0.01; TST, F3,44 = 6.006, P = 0.0016; OFT, F3,44 = 4.677, P = 0.0064; EPM, F3,44 = 4.489, P = 0.0078). d, e,Typical traces of 5-HT detected by electrochemical detection combined with HPLC (d) and summarized data of 5-HT concentration in the CeA from the indicated groups (e, n = 8 mice each group; F3,28 = 3.525, P = 0.0277). f, Summarized data of the resting membrane potential (Vrest) recorded from SOMCeA neurons in the indicated groups (Sham 6W, n = 12 cells; SNI 6W, n = 15 cells; t25 = 1.524, P = 0.1401). Significance was assessed by two-way RM ANOVA with post hoc comparison between groups in a, two-tailed unpaired Student’s t test in b, f, and ordinary one-way ANOVA with post hoc comparison between groups in c, e. The data are expressed as the mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001. ns, no significance.

Supplementary Figure 5 Inhibition of the 5-HTDRN→CeA pathway produces depressive-like behaviors.

a, Behavioural effects of optogenetic activation of 5-HTDRN terminals in the CeA in response to WAY (mCherry + ASCF and mCherry + WAY, n = 10 mice per group; ChR2 + ACSF and ChR2 + WAY, n = 9 mice per group; F3,34 = 15.83, P < 0.0001; pre vs light post hoc comparison, mCherry + ASCF, P > 0.9; ChR2 + ACSF, P < 0.0001; ChR2 + WAY, P > 0.9; mCherry + WAY, P = 0.3947). b, Pain threshold in Pet1-Cre mice with DRN infusion of AAV-DIO-ChR2-mCherry (ChR2-mCherry) or control virus (mCherry) after photostimulation in the CeA (n = 9 mice per group; F2,32 = 5.03, P = 0.0126). c, Schematic of viral injection and optical manipulation. d, Sample trace (left) and statistical data (right) of currents recorded from AAV-DIO-eNpHR3.0-EYFP-expressing 5-HTDRN neurons via photostimulation (594 nm) in acute slices from Pet1-Cre mice (n = 9 cells, one sample t test, t8 = −12.564, P < 0.0001). e, Statistic charts showing the behavioural effects of inhibition of 5-HTDRN terminals in the CeA (EYFP, n = 9 mice; eNpHR3.0-EYFP, n = 10 mice; SPT, t17 = 2.815, P = 0.0119; TST, F1,17 = 24.35, P = 0.0001; pre vs light post hoc comparison, EYFP, P = 0.1810, eNpHR3.0-EYFP, P = 0.0001). Significance was assessed by two-way RM ANOVA with post hoc comparison between groups in a, b, e (TST), and two-tailed unpaired Student’s t test in e (SPT). The data are expressed as the mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001. ns, no significance.

Supplementary Figure 6 Behavioural effects of activation of the 5-HTDRN→SOMCeA circuit in mice treated with chronic stress.

a, Performance of chronic restraint stress (CRS) and control (CON) mice in SPT, TST, OFT, and EPM (n = 10 per group; SPT, t18 = 2.669, P = 0.0188; TST, t18 = 2.865, P = 0.0103; OFT, t18 = 2.366, P = 0.0322; EPM, t18 = 2.375, P = 0.0289). b, Summarized data of 5-HT concentration in the CeA from CRS and CON mice (n = 6 per group; t10 = 0.6872, P = 0.5076). c, Performance of social defeat stress (SDS, n = 15) and control (CON, n = 10) mice in SPT, TST, OFT, and EPM (SPT, t23 = 3.139, P = 0.0046; TST, t23 = 2.603, P = 0.0159; OFT, t23 = 5.282, P < 0.0001; EPM, t23 = 4.354, P = 0.0002). d, Summarized data of 5-HT concentration in the CeA from SDS or CON mice (n = 6 per group; t10 = 0.5819, P = 0.5735). e, Statistical data showing the effect of optical activation of 5-HTDRN terminals in the CeA in CRS mice (CRS + mCherry, n = 10 mice; CRS + ChR2-mCherry, n = 8 mice; SPT, t16 = 0.5517, P = 0.5888; TST, F2,32 = 1.661, P = 0.2059). f, Statistical data showing the effect of optical activation of 5-HTDRN terminals in the CeA in SDS mice. (n = 8 per group; SPT, t16 = 0.1535, P = 0.8802; TST, F2,28 = 0.474, P = 0.6274). Significance was assessed by two-tailed unpaired Student’s t test in a-d, e (SPT), f (SPT), and two-way RM ANOVA with post hoc comparison between groups in e (TST), f (TST). The data are expressed as the mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001. ns, no significance.

Supplementary Figure 7 Outputs of SOMCeA neurons.

a, Representative images of mCherry expression in SOM-Cre mice after CeA injection of AAV-DIO-ChR2-mCherry. Scale bars: 300 µm (left), 50 µm (right). bf, mCherry signals in the lateral periaqueductal gray matter (LPAG, b), the nucleus accumbens (AcbC, c), the medial geniculate nucleus (MG, d), the bed nucleus of the stria terminalis (BNST, e), and the substantia nigra pars reticulata (SNR, f). Scale bars: 500 µm (left), 50 µm (right). The experiment was independently repeated five times with similar results obtained.

Supplementary Figure 8 Activation of the SOMCeA→LHb pathway produces depressive-like behaviours.

a, Schematic of the Cre-dependent retrograde trans-monosynaptic rabies virus tracing strategy. b, Representative images of viral expression within the LHb of the GAD2-Cre mice (left). No DsRed signal was observed in the CeA (right). The experiment was independently repeated with similar results in n = 5 mice. Scale bars: 100 µm (left), 200 µm (right). c,d, Representative images (c) and percentage (d) of DsRed signals from the LHb co-localized with 5-HT1ARs in the CeA (n = 9 slices from three mice). Scale bar: 50 µm. e, Representative images and percentage of DsRed signals from the LHb co-localized with 5-HT2ARs in the CeA (n = 9 slices from three mice). Scale bar: 50 µm. f, Schematic of viral injection for CeA-LHb tracing. g,h, Typical images of GFP expression in the LHb of mice with CeA infusion of AAV2/1-hSyn-Cre (g, Scale bars: 300 µm left, 50 µm right), which co-localized with VGLUT2 (h, Scale bar: 50 µm). The experiment was independently repeated with similar results in n = 9 slices from three mice. i, Schematic of viral injection and optical manipulation in the LHb. j, Behavioural effects of photostimulation in the LHb of SOM-Cre mice with CeA injection of AAV-DIO-ChR2-mCherry or control virus (ChR2-mCherry, n = 10 mice; mCherry, n = 8 mice; SPT, t16 = 2.285, P = 0.0398; TST, F2,32 = 3.572, P = 0.0398; pre vs light post hoc comparison, ChR2-mCherry, P = 0.0004; mCherry, P > 0.9). k, Behavioural effects of optical inhibition of SOMCeA terminals in the LHb of SNI 6W SOM-Cre mice with CeA injection of AAV-DIO-eNpHR3.0-EYFP (eNpHR3.0-EYFP) or control virus EYFP (eNpHR3.0-EYFP, n = 9; EYFP, n = 10; F2,34 = 7.809, P = 0.0016; pre vs light post hoc comparison, eNpHR3.0-EYFP, P < 0.0001; EYFP, P > 0.9). For d, e and h, data are shown as box and whisker plots [medians, quartiles (boxes) and ranges min to max (whiskers)]. Significance was assessed by two-tailed unpaired Student’s t test in j (SPT), and two-way RM ANOVA with post hoc comparison between groups in j (TST), k. The data are expressed as the mean ± s.e.m. *P < 0.05; ***P < 0.001. ns, no significance.

Supplementary Figure 9 A model of the disinhibitory 5-HTDRN→SOMCeA→GluLHb circuit in CDS.

In the chronic pain state, a disinhibitory process occurs within the CeA, which involves decreased 5-HTDRN inputs that mediate inhibition of SOMCeA neurons (red) through 5-HT1AR, but mediate excitation of non-SOM interneurons (NSCeA, green) through 5-HT2AR. Both alterations lead to even higher excitation of SOMCeA neurons. As a result, the SOMCeA glutamatergic outputs (red) to the LHb are enhanced, which presumably causes CDS. DRN, dorsal raphe nucleus; LHb, lateral habenula; CeA, central nucleus of the amygdala; 5-HT, 5-hydroxytryptamine; 5-HT1ARs, 5-HT1A receptors; 5-HT2ARs, 5-HT2A receptors; GABA, gamma-aminobutyric acid; Glu, glutamate; AMPARs, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors.

Supplementary Figure 10 Altered functional connectivity with the right CM in CDS patients.

a, Location of the subregions of the human right amygdala. Red, centromedial amygdala (CM); green, laterobasal nuclei; blue, superficial nuclei. b,c, fMRI images showing brain areas with significant differences in functional connectivity with the right CM in CDS patients (n = 22) compared with depressed patients without chronic pain (b, n = 27) and healthy controls (c, n = 27). d,e, Brain areas showing altered functional connectivity with the right CM in depressed patients without chronic pain (d) and chronic pain patients without depression (e, n = 29) compared with healthy controls. f, Statistical values of CM-DRN functional connectivity in the indicated groups (F3,101 = 13.72, P < 0.0001). g, The Pearson correlation analysis of the functional connectivity of the DRN-CM pathway and the HAMD score in CDS and MDD patients (CDS, P = 0.0169, r = -0.5036; MDD, P = 0.2029, r = -0.253). h, Summarized data of the HAMD scores of CDS and MDD patients (t47 = 0.9976, P = 0.3236). MDD, major depression disorder without pain; CP, chronic pain without depression. The numbers beneath the images refer to the coordinates in the Montreal Neurological Institute space. The red circles in each image represent the regions that contain the DRN. The t-score bars are shown at the right of images. Significance was assessed by one-way ANOVA with post hoc comparison between groups in f, and two-tailed unpaired Student’s t test in h. The data are expressed as the mean ± s.e.m. **P < 0.01; ***P < 0.001. ns, no significance.

Supplementary Figure 11 Altered functional connectivity with the left CM in CDS patients.

ad, fMRI images showing brain areas with significant differences in functional connectivity with the amygdala compared with the indicated groups. eh, fMRI images showing brain areas with significant differences in functional connectivity with the left CM compared with the indicated groups. The numbers beneath the images refer to the coordinates in the Montreal Neurological Institute space. The red circles in each image represent the regions that contain the DRN. The t-score bars are shown at the right of images.

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Zhou, W., Jin, Y., Meng, Q. et al. A neural circuit for comorbid depressive symptoms in chronic pain. Nat Neurosci 22, 1649–1658 (2019). https://doi.org/10.1038/s41593-019-0468-2

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