Brief Communication | Published:

Morphine withdrawal recruits lateral habenula cytokine signaling to reduce synaptic excitation and sociability

Abstract

The lateral habenula encodes aversive stimuli contributing to negative emotional states during drug withdrawal. Here we report that morphine withdrawal in mice leads to microglia adaptations and diminishes glutamatergic transmission onto raphe-projecting lateral habenula neurons. Chemogenetic inhibition of this circuit promotes morphine withdrawal-like social deficits. Morphine withdrawal-driven synaptic plasticity and reduced sociability require tumor necrosis factor-α (TNF-α) release and neuronal TNF receptor 1 activation. Hence, habenular cytokines control synaptic and behavioral adaptations during drug withdrawal.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request.

References

  1. 1.

    Goeldner, C. et al. Biol. Psychiatry 69, 236–244 (2011).

  2. 2.

    Lutz, P. E. et al. Neuropsychopharmacology 39, 2694–2705 (2014).

  3. 3.

    Meye, F. J., Trusel, M., Soiza-Reilly, M. & Mameli, M. Pharm. Biochem. Behav. 162, 87–93 (2017).

  4. 4.

    Margolis, E. B. & Fields, H. L. PLoS One 11, e0159097 (2016).

  5. 5.

    Wang, J. et al. Neurosci. Lett. 653, 64–70 (2017).

  6. 6.

    Valentinova, K. & Mameli, M. Cell Rep. 16, 2298–2307 (2016).

  7. 7.

    Pollak Dorocic, I. et al. Neuron 83, 663–678 (2014).

  8. 8.

    Lewitus, G. M. et al. Neuron 90, 483–491 (2016).

  9. 9.

    Campbell, L. A., Avdoshina, V., Rozzi, S. & Mocchetti, I. Brain Behav. Immun. 34, 130–140 (2013).

  10. 10.

    Michaud, M. et al. J. Am. Med. Dir. Assoc. 14, 877–882 (2013).

  11. 11.

    Probert, L. Neuroscience 302, 2–22 (2015).

  12. 12.

    Nie, X. et al. Neuron 99, 464–479.e7 (2018).

  13. 13.

    Hutchinson, M. R. et al. Brain Behav. Immun. 22, 1178–1189 (2008).

  14. 14.

    Patel, A., Siegel, A. & Zalcman, S. S. Brain Behav. Immun. 24, 1276–1280 (2010).

  15. 15.

    He, P., Liu, Q., Wu, J. & Shen, Y. FASEB J. 26, 334–345 (2012).

  16. 16.

    Stellwagen, D., Beattie, E. C., Seo, J. Y. & Malenka, R. C. J. Neurosci. 25, 3219–3228 (2005).

  17. 17.

    Kane, M. J. et al. PLoS One 7, e48975 (2012).

  18. 18.

    Lecca, S. et al. eLife 6, e30697 (2017).

  19. 19.

    Matthews, G. A. et al. Cell 164, 617–631 (2016).

Download references

Acknowledgements

The authors thank F.J. Meye and the Mameli Laboratory for comments on the manuscript. This work was supported by funds from the ERC StG SalienSy 335333 and the SNSF (31003A) to M.M. They also thank D. Szymkowski for the donation of XENP1595, and G. Kollias and H. Strubbe for the use and breeding of the Tnfr1fl/fl mouse line.

Author information

K.V., A.T. and M.M. performed and analyzed the ex vivo recordings and behavior experiments. A.L.L. and J.A.C. contributed to the ex vivo recordings. M.T., I.M. and L.M. performed the molecular biology experiments. C.B. and S.T. provided support for the behavioral experiments. A.M. and R.C.P. analyzed the microglia morphology. A.V. provided conceptual and experimental input related to TNF-α signaling and the Tnfr1fl/fl mice. K.V. and M.M. conceptualized, designed the study and wrote the manuscript.

Correspondence to Manuel Mameli.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Journal peer review information: Nature Neuroscience thanks David Stellwagen and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Integrated supplementary information

Supplementary Figure 1 MORwd induces postsynaptic depression of AMPAR-mediated neurotransmission in medial LHb.

(a) Left: sample traces, box and scatter plots of sEPSCs amplitudes recorded in LatLHb (saline+naloxone (ncells/mice = 9/3; gray) versus NP-MORwd (ncells/mice = 9/4; orange), two-sided t-test,, t16 = 0.098, P = 0.923). Right: same but sEPSCs were recorded in MedLHb (saline+naloxone (ncells/mice = 9/4; black) versus NP- MORwd (ncells/mice = 9/5; red), two-sided t-test, t16 = 3.493, **P = 0.003). (b) Left: sample traces, box and scatter plots of sEPSCs frequencies recorded in LatLHb (saline+naloxone (ncells/mice = 9/4, gray) versus NP-MORwd (ncells/mice = 9/4, orange), two-sided t-test,, t16 = 1.331, P = 0.202). Right: same but sEPSCs were recorded in MedLHb (saline+naloxone (ncells/mice = 9/5, black) versus NP-MORwd (ncells/mice = 9/4,red), two-sided t-test, t16 = 0.161, P = 0.874). (c) Recording map color-coded for the value of AMPAR:NMDAR ratios recorded throughout the LHb. Lighter colors indicate smaller AMPAR:NMDAR ratios, while darker colors represent high AMPAR:NMDAR ratio. (d) Top: Sample traces and normalized EPSC versus pulse number plots recorded at 5, 10 and 20 Hz in LatLHb (saline+naloxone (ncells/mice = 10/2, gray) versus NP-MORwd (ncells/mice = 10/3, orange), 5 Hz interaction factor F(4, 36) = 0.227, P = 0.921; 10 Hz interaction factor F(4,36) = 0.251, P = 0.907; 20Hz interaction factor F(4,36) = 0.573, P = 0.683 two-way ANOVA Repeated Measures). Bottom: same but in MedLHb (ncells/mice = 10/3, saline+naloxone (black) versus NP- MORwd (red), 5Hz interaction factor F(4,36) = 0.1.183, P = 0.334; 10Hz interaction factor F(4,36) = 1.171, P=0.34; 20Hz interaction factor F(4,36) = 0.88, P = 0.485 two-way ANOVA Repeated Measures). (e) Spontaneous withdrawal timeline. AMPAR:NMDAR ratios from MedLHb 1 h, 10, 20 or 30 days post-saline or MOR (saline 1 h and 10 days pooled (nmice/cells = 6/22; black) versus MORwd 1 h (nmice/cells = 5/11; open red) and MOR 10 (nmice/cells = 3/12), 20 (nmice/cells = 3/11) and 30 days withdrawal (nmice/cells = 3/11; red), F(4, 62) = 3.90 one-way ANOVA, **P = 0.007). (f) Example of peak-scaled NSFA of MedLHb neurons in the saline- and NP-MORwd group. Pooled data for conductance (γ) and number of channels (N) open at the peak together with amplitude versus N of channels and conductance plots (Saline+naloxone, ncells/mice = 5/4; MORwd, ncells/mice = 8/5; N of channels, two-sided t-test, t11 = 5.67, ***P = 0.0001, r2(N-Channels) = 0.416; *P = 0.017; Conductance, t11 = 0.006, P = 0.99, r2(Conductance) = 0.03, P = 0.55). (g) Left: sample traces, box and scatter plots for rectification index calculated from AMPAR EPSCs recorded at -70, 0 and 40 mV in LatLH (saline+naloxone (ncells/mice = 9/7, gray) versus NP- MORwd (ncells/mice = 10/7, orange), two-sided t-test, t17 = 0.210, P = 0.836). Right same but recordings in MedLHb (saline+naloxone (ncells/mice = 12/8, black) versus NP- MORwd (ncells/mice = 9/5, red), two-sided t-test, t19 = 1.292, P = 0.212). (h) Sample traces, box and scatter plots of AMPAR:NMDAR ratios recorded in MedLHb via 405 nm laser-assisted uncaging of MNI-glutamate, 500 μM (saline+naloxone (ncells/mice = 8/2, black) versus NP-MORwd (ncells/mice = 10/3, red), two-sided t-test, t16 = 3.521, **P = 0.003). Bottom right: Absolute AMPAR versus absolute NMDAR uncaging-evoked current plots from saline+naloxone (open black circles) or NP-MORwd mice (open red circles). The mean with S.E.M. AMPA and NMDA currents are shown with black and red filled circles for saline versus MORwd respectively (saline+naloxone versus NP-MORwd: AMPA, two-sided t-test, t16 = 3.536, **P = 0.003; NMDA, two-sided t-test, t16 = 0.195, P = 0.848). Data are presented as box plots 10–90 percentiles and scatter.

Supplementary Figure 2 TNFα levels in the LHb increase following spontaneous MORwd.

(a) TNFα (cyan) and DAPI (magenta) immunostaining in slices from saline-treated, (b) MOR-treated (sacrificed 1 h after the last MOR injection) and (c) animals in spontaneous MORwd (10–13 days post last MOR injection). (d) Normalized LHb TNFα optical density in saline (black), MOR (open red) and spontaneous MORwd (red) (nmice = 8, saline (black) versus MOR (open red) versus spontaneous MORwd (red), F(2,20) = 7.7 one-way ANOVA, **P = 0.003). Data are presented as box plots 10–90 percentiles with median and scatter.

Supplementary Figure 3 TNFα signaling is necessary and sufficient for MORwd-induced plasticity.

(a) Spontaneous MORwd protocol, sample traces, box and scatter plots for AMPAR:NMDAR ratios recorded in MedLHb slices incubated with (+) or without (–) exogenous TNFα from spontaneous MORwd mice (10 days). (MORwd -TNFα (red) versus +TNFα (pink), ncells/mice = 9/4, two-sided t-test, t16 = 0.986, P = 0.339). (b) MedLHb AMPAR:NMDAR ratios from saline or MPLA-injected MOR-treated mice (MOR/saline (nmice/cells = 3/10; shaded blue) versus MOR/MPLA (nmice/cells = 4/11; dark blue), two-sided t-test, t19 = 3.070, **P = 0.006). (c) Sample traces, time versus amplitude plot and bar graphs showing the effect of MPLA (1 μg/ml) on AMPAR-EPSCs (MOR (nmice/cells = 4/11 ; 63.93 ± 7.06%; open red), NP-MORwd (nmice/cells = 3/10 cells; 91.63 ± 8.86%; filled red), MOR versus NP-MORwd, two-sided t-test, t19 = 2.419, *P = 0.026). Data of this panel are represented as mean and sem. (d) Sample traces, time versus amplitude plot and bar graphs showing the effect of MPLA (1 μg/ml) on evoked AMPAR-EPSCs (baseline (1) vs 30 min post-MPLA (2)) recorded in LatLHb (open orange) or MedLHb (open red) in slices obtained from morphine-treated animals (ncells/mice=8/4, morphine LatLHb 103.98 ± 10.13; ncells/mice = 11/5, morphine MedLHb 63.31 ± 7.06%; morphine LatLHb versus morphine MedLHb, two-sided t-test, t17 = 3.406, **P = 0.003). Note that the data set for MedLHb is the same as in c and is used for comparison. Data are presented as mean and SEM. (e) Sample traces, time versus amplitude plot and bar graph showing the effect of MPLA (1 μg/ml) on evoked AMPAR-EPSCs (baseline (1) vs 30 min post-MPLA (2)) in the presence of TNFα dominant negative peptide (XENP1595, 6mg/1ml) recorded in MedLHb in slices obtained from morphine-treated animals (ncells/mice = 7/2, 98.88 ± 8.23 %, two-sided t-test, t-test, t6 = 0.073, P = 0.944). Data are presented with mean and SEM. (f) NP-MORwd protocol with dominant-negative TNFα (XENP1595, 30 mg/kg) pretreatment, sample traces, box and scatter plots for AMPAR:NMDAR ratios recorded in MedLHb (saline+naloxone (ncells/mice = 10/3, black) versus NP-MORwd (ncells/mice = 12/3, green), two-sided t-test, t-test, t20 = 0.165, P = 0.871). Data are presented as box plots 10–90 percentiles and scatter.

Supplementary Figure 4 Behavioral assessment of MORwd.

(a) Box and scatter plot showing the percent time spent in the compartment containing the social stimulus for C57Bl6 mice (N = 22 mice/group, saline+naloxone (black) versus NP-MORwd (red), two-sided t-test, t42 = 2.401, *P = 0.021). (b) Box and scatter plot showing the percent time spent in the compartment containing the object stimulus for C57Bl6 mice (N = 22 mice/group, saline+naloxone (black) versus NP-MORwd (red), two-sided t-test, t42 = 2.465, *P = 0.02). (c) Box and scatter plot showing the percent time spent in the central compartment for C57Bl6 mice (N = 22 mice/group, saline+naloxone (black) versus NP-MORwd (red), two-sided t-test, t42 = 1.186, P = 0.242). (d) Box and scatter plot showing locomotor activity during social preference test for C57Bl6 mice (N = 22 mice/group, saline+naloxone (black) versus NP-MORwd (red), two-sided t-test, t42 = 2.621, *P = 0.012). (e) Box and scatter plot showing the percent time spent in the compartment containing the social stimulus for TNF-R1fl/fl mice (AAV-Control: 58.02 ± 2.96% saline+naloxone (Nmice = 20, black) versus 40.45 ± 5.08% NP-MORwd (Nmice = 23, red); AAV-Cre: 54.41 ± 3.15% saline (Nmice = 13, open gray) versus 64.52 ± 5.94 % NP-MORwd (Nmice = 13, open pink), interaction factor F(1,65) = 8.591 two-way ANOVA, **P = 0.005). (f) Box and scatter plot showing the percent time spent in the compartment containing the object stimulus for TNF-R1fl/fl mice (N of mice same as panel e. AAV-Control: 26.42 ± 2.47 % saline+naloxone (black) versus 30.9 ± 4.79% NP-MORwd (red); AAV-Cre: 31.19 ± 2.07% saline+naloxone (open gray) versus 20.7 ± 4.13 % NP-MORwd (open pink), interaction factor F(1,65) = 4.136 two-way ANOVA, *P = 0.046). (g) Box and scatter plot showing the percent time spent in the central compartment for TNF-R1fl/fl mice (N of mice same as panel e. AAV-Control: 15.38 ± 3.08% saline+naloxone (black) versus 27.82 ± 4.98% NP-MORwd (red); AAV-Cre: 14.23 ± 2.9 % saline+naloxone (open gray) versus 14.62 ± 3.08% NP-MORwd (open pink), interaction factor F(1,65) = 1.748 two-way ANOVA, P = 0.191). (h) Box and scatter plot showing number of exploration bouts of TNF-R1fl/fl with the juvenile (N of mice same as panel e. AAV-Control: 56 ± 3.51 saline+naloxone (black) versus 39.30 ± 4.8 NP-MORwd (red); AAV-Cre: 50.38 ± 3.78 saline+naloxone (open gray) versus 57.15 ± 7.01 NP-MORwd (open pink), interaction factor F(1.65) = 5.519 two-way ANOVA, *P = 0.022). (i) Box and scatter plot showing number of exploration bouts of TNF-R1fl/fl with the object (N of mice is the same as panel e. AAV-Control: 26.9 ± 1.88 saline+naloxone (black) versus 23.35 ± 2.13 NP-MORwd (red); AAV-Cre: 28.08 ± 2.01 saline+naloxone (open gray) versus 21.69 ± 3.16 NP-MORwd (open pink), interaction factor F(1.65) = 0.361, P = 0.55). (j) Box and scatter plot showing locomotor activity during social preference test for TNF-R1fl/fl mice (N of mice is the same as panel e. AAV-Control: 28.13 ± 1.35 m saline+naloxone (black) versus 33.8 ± 5.59 m NP-MORwd (red); AAV-Cre: 23.78 ± 1.47m saline+naloxone (open gray) versus 40.17 ± 7.11 m NP-MORwd (open pink), interaction factor F(1,65) = 1.224 two-way ANOVA, P = 0.273). Data are presented as box plots 10–90 percentiles with median and scatter.

Supplementary information

Supplementary Figures 1–4.

Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Further reading

Fig. 1: MORwd-driven projection-specific synaptic depression in the LHb.
Fig. 2: Cytokine signaling in the LHb for MORwd plasticity.
Fig. 3: TNF-R1 requirements for MORwd-driven synaptic and behavioral adaptations.
Supplementary Figure 1: MORwd induces postsynaptic depression of AMPAR-mediated neurotransmission in medial LHb.
Supplementary Figure 2: TNFα levels in the LHb increase following spontaneous MORwd.
Supplementary Figure 3: TNFα signaling is necessary and sufficient for MORwd-induced plasticity.
Supplementary Figure 4: Behavioral assessment of MORwd.