Abstract
The paraventricular nucleus of the thalamus (PVT) is increasingly being recognized as a critical node linking stress detection to the emergence of adaptive behavioral responses to stress. However, despite growing evidence implicating the PVT in stress processing, the neural mechanisms by which stress impacts PVT neurocircuitry and promotes stressed states remain unknown. Here we show that stress exposure drives a rapid and persistent reduction of inhibitory transmission onto projection neurons of the posterior PVT (pPVT). This stress-induced disinhibition of the pPVT was associated with a locus coeruleus-mediated rise in the extracellular concentration of dopamine in the midline thalamus, required the function of dopamine D2 receptors on PVT neurons, and increased sensitivity to stress. Our findings define the locus coeruleus as an important modulator of PVT function: by controlling the inhibitory tone of the pPVT, it modulates the excitability of pPVT projection neurons and controls stress responsivity.
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Acknowledgements
We thank T. Davidson (Stanford University) for his assistance in the design of the fiber photometry system and G. Augustine (Lee Kong Chian School of Medicine, Singapore) for the SuperClomeleon plasmid. This work was supported by the NIMH Intramural Research Program (M.A.P.), NICHD Intramural Research Program (A.B.), and NIH Grant MH107460 (H.-B.K.).
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B.S.B. performed anatomical and immunohistochemical studies, calcium and chloride imaging experiments, optogenetic experiments, and stereotaxic injections for all experiments. B.J.W. and M.A.P. performed electrophysiological experiments. M.S. performed microdialysis experiments and analyzed collected fractions via HPLC. Y.L. performed RT-PCR and in situ hybridization experiments. J.H.H. performed all procedures for two-photon imaging of gephyrin puncta. O.K. and N.R. performed stereotaxic surgeries and histological procedures. A.B. and H.-B.K. provided critical reagents and suggestions. B.S.B., B.J.W., and M.A.P. designed the study, interpreted results, and wrote the paper.
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Supplementary Figure 1 Inhibitory projections to the pPVT.
Compilation of data from the Mouse Brain Connectivity Atlas of the Allen Brain Institute (connectivity.brain-map.org) showing eight different areas of the mouse brain where a Cre-dependent AAV expressing GFP was injected (left panels), and fluorescently-labelled terminals were detected in the pPVT (right panels). Abbreviations: MPO, medial preoptic area; SI, substantia innominata; AHN, anterior hypothalamic nucleus; DMH, dorsomedial hypothalamus; ZI, zona incerta; PAG, periaqueductal gray; MRN, median raphe nucleus; PRNo, pontine reticular nucleus oral part; PRNc, pontine reticular nucleus caudal part. The following mouse Cre-lines were used for these experiments: GAD2-IRES-Cre (MPO, SI, AHN, DMH, ZI, PAG), Slc32a1-IRES-Cre (MRN/PRNo), and Slc6a5-Cre (PRNc).
Supplementary Figure 2 Most neurons of the pPVT express dopamine D2 receptors.
Representative images showing coincident expression of D2-EGFP and the neuronal marker NeuN in the pPVT of wildtype mice (colocalization: 70% D2+/NeuN, n = 3 mice).
Supplementary Figure 3 Sex differences do not account for stress-induced disinhibition of projection neurons of pPVT.
Quantification of mIPSC amplitude and frequency analyzed across both sexes (blue- males; pink- females). Amplitude in pA, naïve male, 49.93 ± 3.62, n = 17 neurons, 2 mice; naïve female, 49.08 ± 4.83, n = 14 neurons, 2 mice; two-sided t-test, non-significant, P = 0.89; restraint male, 39.05 ± 4.66, n = 7 neurons, 2 mice; restraint female, 36.64 ± 1.07, n = 17 neurons, 2 mice; two-sided t-test, non-significant, P = 0.48; footshock male, 41.93 ± 2.26, n = 21 neurons, 2 mice; footshock female, 40.76 ± 1.83, n = 30 neurons, 2 mice; two-sided t-test, non-significant, P = 0.69. Frequency in Hz, naïve male, 5.51 ± 1.06, n = 17 neurons; naïve female, 4.34 ± 0.73, n = 14 neurons, 2 mice; two-sided t-test, non-significant, P = 0.39; restraint male, 5.33 ± 1.33, n = 7 neurons, 2 mice; restraint female, 2.81 ± 0.39, n = 17 neurons, 2 mice; two-sided t-test, *P = 0.023; footshock male, 5.32 ± 0.89, n = 21 neurons, 2 mice; footshock female, 5.21 ± 0.92, n = 30 neurons, 2 mice; two-sided t-test, non-significant, P = 0.93. Data shown as mean ± s.e.m.
Supplementary Figure 4 SuperClomeleon reports GABAA receptor activity.
a. Schematic of the experimental design used for fiber photometry chloride imaging of NAc-projecting pPVT neurons. b. Representative image of SuperClomeleon expression in NAc-projecting neurons of the pPVT. c. Average SuperClomeleon response from NAc-projecting pPVT neurons in animals injected with the GABAA receptor agonist 4,5,6,7-tetrahydroisoxazolo(5,4-c) pyridin-3-ol (THIP, 8 mg/kg; green line) or saline (gray line; n = 3 mice). Data shown as mean ± s.e.m.
Supplementary Figure 5 Optical fiber placements for fiber photometry and optogenetic experiments.
a. Optical fiber placements for GCaMP6s experiment in Fig. 6a-d. b. Optical fiber placement for GCaMP6s experiment in Fig. 6e-h. c. Optical fiber placements for GCaMP6s experiment in Fig. 6i-l. d. Optical fiber placement for GCaMP6s experiment in Fig. 4h. e. Optical fiber placement for GCaMP6s experiment in Supplementary Fig. 8. f. Optical fiber placements for SuperClomeleon experiment in Fig. 1e-h. g. Optical fiber placement for SuperClomeleon experiment in Fig. 5a-d. h. Optical fiber placements for SuperClomeleon experiment in Fig. 5e-h. i. Optical fiber placement for SuperClomeleon experiment in Supplementary Fig. 3. j. Optical fiber placement for ChR2 experiment in Fig. 7. k. Optical fiber placement for halorhodopsin experiment in Fig. 6e-h. All circles depict the lowest position of the optical fibers for each subject.
Supplementary Figure 6 Robust expression of Drd2 and weak expression of Drd3 in the pPVT.
a. Fluorescent 2-plex in situ hybridization experiment showing the expression of Drd2 (red) and Drd3 (green) mRNA in the pPVT. b. Magnified view of the region depicted by the white square (a). 2-plex in situ hybridization experiments were independently repeated three times with samples collected from different subjects and similar results were obtained.
Supplementary Figure 7 Probe placements for microdialysis experiments.
a. Probe placements for microdialysis experiment in Fig. 4b. b. Representative image of hM4Di-mCherry expression in the LC of Dbh-Cre mice used for combined chemogenetic silencing of the LC and microdialysis of the PVT. Histological assessment of hM4Di-mCherry expression in the LC was independently repeated for each mouse included (seven total) and similar results were obtained. c. Probe placements for microdialysis experiment in saline treated mice (Fig. 4j). d. Probe placements for microdialysis experiment in CNO treated mice (Fig. 4j).
Supplementary Figure 8 TH+ afferents of the pPVT originate in the LC.
a. Representative image showing viral-assisted retrograde labelling of pPVT-projecting neurons in the ventral tegmental area (VTA). Antibody staining reveals TH-expressing neurons in VTA. Histological assessment of TH-expressing PVT-projecting neurons in the VTA was independently repeated for two mice and similar results were obtained. b. Magnified portion of the image shown in (a) depicts a single retrogradely-labelled neuron in VTA (arrow) that does not appear to be immunopositive for TH. c. Representative images from two separate experiments of viral-assisted retrograde labelling of pPVT-projecting neurons in the ventral tegmental area in LC. All neurons located within the LC displayed TH immunoreactivity. Histological assessment of TH-expressing PVT-projecting neurons in the LC was independently repeated for two mice and similar results were obtained.
Supplementary Figure 9 Stress-induced increase in extracellular NE in the pPVT is only partially dependent on the LC.
Schematic of the approach utilized for combined chemogenetic silencing of the LC and microdialysis of the pPVT (left). Summary plot depicting stress-induced increases in the extracellular concentration of NE following CNO (black) and saline vehicle (red) I.P. injection in mice expressing hM4Di in LC (n = 7 mice, per group). Data shown as mean ± s.e.m.
Supplementary Figure 10 Pharmacological blockade of the GABAA receptor increases the firing rate of neurons of the pPVT.
a. Sample traces showing the effect of the GABAA receptor blocker picrotoxin (PTX; 100 µM) on cell-attached recorded action potentials from two different pPVT neurons. b. Quantification of firing frequency in Hz (Baseline, 0.86 ± 0.23; PTX, 1.67 ± 0.41; n = 9 neurons, 3 mice, **P = 0.0082; two-sided Paired sample t-test). Data shown as mean ± s.e.m.
Supplementary Figure 11 Stress activates D2+ neurons of the pPVT.
a. Schematic of viral vector injections and optical fiber implantation for GCaMP6 fiber photometry experiments in Drd2-Cre mice. b. Representative image of GCaMP6s expression in D2+ neurons of the pPVT and optical fiber placement. Histological assessment of GCaMP6s expression in D2+ neurons of the pPVT was independently repeated for each mouse included (four total) and similar results were obtained. c. Average GCaMP6s response from D2+ pPVT neurons in animals subjected to footshock stress. Individual footshocks depicted by arrowheads. d. Average change in baseline fluorescence following footshock stress in %dF/F: Before, −0.68 ± 0.46; After, 6.71 ± 1.94; n = 4 mice; *P = 0.049; two-sided Paired sample t-test. Data shown as mean ± s.e.m.
Supplementary Figure 12 Most NAc-projecting neurons of the pPVT express D2 receptors.
a. Schematic of the stereotaxic injections for selectively expressing CTB in NAc-projecting neurons of the pPVT of D2-EGFP mice. b. Representative images showing coincident expression of D2-EGFP and CTB in the pPVT of wildtype mice (colocalization: 80% double-labelled/CTB, n = 4 mice).
Supplementary Figure 13 Tail suspension stress induces rapid activation of NAc-projecting neurons of the pPVT.
a. Schematic of the tail suspension protocol used during GCaMP6s imaging of NAc-projecting pPVT neurons. b. Representative trace showing the effect of brief tail suspensions (~10-12 s) on GCaMP6s fluorescence on a single subject. Arrowheads depict individual tail suspension events. c. Average GCaMP6s response from NAc-projecting pPVT neurons in animals subjected to tail suspension. Individual tail suspension events depicted by arrowheads. d. Average change in baseline fluorescence following tail suspension stress in %dF/F: Before, 0.24 ± 0.45; After, 9.88 ± 2.98; n = 4 mice; *P = 0.039; two-sided Paired sample t-test. Data shown as mean ± s.e.m.
Supplementary Figure 14 Changes in GCaMP6s fluorescence do not correlate with movement.
Correlation between the locomotion index (extracted from the FreezeFrame software, Actimetrics) and the GCaMP6s fluorescent signal (in %dF/F) for two individual subjects (n = 2 mice) from data shown in Fig. 6a-d. R-Square values are 0.008 (left) and 0.0004 (right).
Supplementary Figure 15 Optogenetic stimulation of LC–pPVT projections drives D2-receptor-mediated disinhibition in vitro.
a. Schematic of the experimental procedure employed to assess the effect of optogenetic stimulation of LC terminals onto NAc-projecting neurons of the pPVT. To elicit ChR2-evoked responses in the presence of TTX, 4-aminopyridine (4-AP, 100 µM) was added to the bath. b. Average plot depicting the effect of optogenetic stimulation of LC terminals with ChR2 on the average amplitude of mIPSC recorded from CTB-positive NAc-projecting neurons in the presence (% of Baseline, n = 9 neurons, 5 mice) or absence (% of Baseline, n = 8 neurons, 4 mice) of the D2-like blocker sulpiride (1 µM). c. Average plot depicting the effect of optogenetic stimulation of LC terminals with ChR2 on the average frequency of mIPSC recorded from CTB-positive NAc-projecting neurons in the presence (% of Baseline, n = 9 neurons, 5 mice) or absence (% of Baseline, n = 8 neurons, 4 mice) of the D2-like blocker sulpiride. Data shown as mean ± s.e.m.
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Beas, B.S., Wright, B.J., Skirzewski, M. et al. The locus coeruleus drives disinhibition in the midline thalamus via a dopaminergic mechanism. Nat Neurosci 21, 963–973 (2018). https://doi.org/10.1038/s41593-018-0167-4
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DOI: https://doi.org/10.1038/s41593-018-0167-4
