Ketamine blocks bursting in the lateral habenula to rapidly relieve depression

Abstract

The N-methyl-d-aspartate receptor (NMDAR) antagonist ketamine has attracted enormous interest in mental health research owing to its rapid antidepressant actions, but its mechanism of action has remained elusive. Here we show that blockade of NMDAR-dependent bursting activity in the ‘anti-reward center’, the lateral habenula (LHb), mediates the rapid antidepressant actions of ketamine in rat and mouse models of depression. LHb neurons show a significant increase in burst activity and theta-band synchronization in depressive-like animals, which is reversed by ketamine. Burst-evoking photostimulation of LHb drives behavioural despair and anhedonia. Pharmacology and modelling experiments reveal that LHb bursting requires both NMDARs and low-voltage-sensitive T-type calcium channels (T-VSCCs). Furthermore, local blockade of NMDAR or T-VSCCs in the LHb is sufficient to induce rapid antidepressant effects. Our results suggest a simple model whereby ketamine quickly elevates mood by blocking NMDAR-dependent bursting activity of LHb neurons to disinhibit downstream monoaminergic reward centres, and provide a framework for developing new rapid-acting antidepressants.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Local blockade of NMDARs in LHb is sufficient to elicit rapid antidepressant effects.
Figure 2: Ketamine suppresses enhanced LHb bursting activity and theta-band synchronization in animal models of depression.
Figure 3: LHb bursting requires activation of NMDARs.
Figure 4: Antagonists of T-VSCCs block LHb bursts and cause rapid antidepression.
Figure 5: eNpHR3.0-induced rebound bursting drives behavioural aversion and depression-like symptoms that are reversible by ketamine.

References

  1. 1

    Berman, R. M. et al. Antidepressant effects of ketamine in depressed patients. Biol. Psychiatry 47, 351–354 (2000)

    Article  CAS  Google Scholar 

  2. 2

    Zarate, C. A. Jr et al. A randomized trial of an N-methyl-d-aspartate antagonist in treatment-resistant major depression. Arch. Gen. Psychiatry 63, 856–864 (2006)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. 3

    Maeng, S. et al. Cellular mechanisms underlying the antidepressant effects of ketamine: role of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors. Biol. Psychiatry 63, 349–352 (2008)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. 4

    Li, N. et al. mTOR-dependent synapse formation underlies the rapid antidepressant effects of NMDA antagonists. Science 329, 959–964 (2010)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  5. 5

    Autry, A. E. et al. NMDA receptor blockade at rest triggers rapid behavioural antidepressant responses. Nature 475, 91–95 (2011)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. 6

    Zanos, P. et al. NMDAR inhibition-independent antidepressant actions of ketamine metabolites. Nature 533, 481–486 (2016)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  7. 7

    Clements, J. A., Nimmo, W. S. & Grant, I. S. Bioavailability, pharmacokinetics, and analgesic activity of ketamine in humans. J. Pharm. Sci. 71, 539–542 (1982)

    Article  CAS  PubMed  Google Scholar 

  8. 8

    Homayoun, H. & Moghaddam, B. NMDA receptor hypofunction produces opposite effects on prefrontal cortex interneurons and pyramidal neurons. J. Neurosci. 27, 11496–11500 (2007)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. 9

    Matsumoto, M. & Hikosaka, O. Lateral habenula as a source of negative reward signals in dopamine neurons. Nature 447, 1111–1115 (2007)

    Article  ADS  CAS  PubMed  Google Scholar 

  10. 10

    Lammel, S. et al. Input-specific control of reward and aversion in the ventral tegmental area. Nature 491, 212–217 (2012)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  11. 11

    Shabel, S. J., Proulx, C. D., Trias, A., Murphy, R. T. & Malinow, R. Input to the lateral habenula from the basal ganglia is excitatory, aversive, and suppressed by serotonin. Neuron 74, 475–481 (2012)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. 12

    Stamatakis, A. M. & Stuber, G. D. Activation of lateral habenula inputs to the ventral midbrain promotes behavioral avoidance. Nat. Neurosci. 15, 1105–1107 (2012)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. 13

    Stephenson-Jones, M. et al. A basal ganglia circuit for evaluating action outcomes. Nature 539, 289–293 (2016)

    Article  ADS  PubMed  PubMed Central  Google Scholar 

  14. 14

    Morris, J. S., Smith, K. A., Cowen, P. J., Friston, K. J. & Dolan, R. J. Covariation of activity in habenula and dorsal raphé nuclei following tryptophan depletion. Neuroimage 10, 163–172 (1999)

    Article  CAS  PubMed  Google Scholar 

  15. 15

    Shumake, J., Edwards, E. & Gonzalez-Lima, F. Opposite metabolic changes in the habenula and ventral tegmental area of a genetic model of helpless behavior. Brain Res. 963, 274–281 (2003)

    Article  CAS  PubMed  Google Scholar 

  16. 16

    Li, B. et al. Synaptic potentiation onto habenula neurons in the learned helplessness model of depression. Nature 470, 535–539 (2011)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  17. 17

    Li, K. et al. βCaMKII in lateral habenula mediates core symptoms of depression. Science 341, 1016–1020 (2013)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  18. 18

    Lecca, S. et al. Rescue of GABAB and GIRK function in the lateral habenula by protein phosphatase 2A inhibition ameliorates depression-like phenotypes in mice. Nat. Med. 22, 254–261 (2016)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. 19

    Aizawa, H., Kobayashi, M., Tanaka, S., Fukai, T. & Okamoto, H. Molecular characterization of the subnuclei in rat habenula. J. Comp. Neurol. 520, 4051–4066 (2012)

    Article  CAS  PubMed  Google Scholar 

  20. 20

    Tye, K. M. et al. Dopamine neurons modulate neural encoding and expression of depression-related behaviour. Nature 493, 537–541 (2013)

    Article  ADS  CAS  Google Scholar 

  21. 21

    Hu, H. Reward and aversion. Annu. Rev. Neurosci. 39, 297–324 (2016)

    Article  CAS  PubMed  Google Scholar 

  22. 22

    Jhou, T. C., Fields, H. L., Baxter, M. G., Saper, C. B. & Holland, P. C. The rostromedial tegmental nucleus (RMTg), a GABAergic afferent to midbrain dopamine neurons, encodes aversive stimuli and inhibits motor responses. Neuron 61, 786–800 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. 23

    Tian, J. & Uchida, N. Habenula lesions reveal that multiple mechanisms underlie dopamine prediction errors. Neuron 87, 1304–1316 (2015)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. 24

    Zhou, L. et al. Organization of functional long-range circuits controlling the activity of serotonergic neurons in the dorsal raphe nucleus. Cell Reports 18, 3018–3032 (2017)

    Article  CAS  PubMed  Google Scholar 

  25. 25

    Chang, S. Y. & Kim, U. Ionic mechanism of long-lasting discharges of action potentials triggered by membrane hyperpolarization in the medial lateral habenula. J. Neurosci. 24, 2172–2181 (2004)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. 26

    Weiss, T. & Veh, R. W. Morphological and electrophysiological characteristics of neurons within identified subnuclei of the lateral habenula in rat brain slices. Neuroscience 172, 74–93 (2011)

    Article  CAS  PubMed  Google Scholar 

  27. 27

    McCormick, D. A. & Bal, T. Sleep and arousal: thalamocortical mechanisms. Annu. Rev. Neurosci. 20, 185–215 (1997)

    Article  CAS  PubMed  Google Scholar 

  28. 28

    Grillner, S., McClellan, A., Sigvardt, K., Wallén, P. & Wilén, M. Activation of NMDA-receptors elicits “fictive locomotion” in lamprey spinal cord in vitro. Acta Physiol. Scand. 113, 549–551 (1981)

    Article  CAS  PubMed  Google Scholar 

  29. 29

    Schiller, J., Major, G., Koester, H. J. & Schiller, Y. NMDA spikes in basal dendrites of cortical pyramidal neurons. Nature 404, 285–289 (2000)

    Article  ADS  CAS  PubMed  Google Scholar 

  30. 30

    Zhu, Z. T., Munhall, A., Shen, K. Z. & Johnson, S. W. NMDA enhances a depolarization-activated inward current in subthalamic neurons. Neuropharmacology 49, 317–327 (2005)

    Article  CAS  PubMed  Google Scholar 

  31. 31

    Cheong, E. & Shin, H. S. T-type Ca2+ channels in normal and abnormal brain functions. Physiol. Rev. 93, 961–992 (2013)

    Article  CAS  PubMed  Google Scholar 

  32. 32

    Huguenard, J. R., Gutnick, M. J. & Prince, D. A. Transient Ca2+ currents in neurons isolated from rat lateral habenula. J. Neurophysiol. 70, 158–166 (1993)

    Article  CAS  PubMed  Google Scholar 

  33. 33

    Lisman, J. E. Bursts as a unit of neural information: making unreliable synapses reliable. Trends Neurosci. 20, 38–43 (1997)

    Article  CAS  PubMed  Google Scholar 

  34. 34

    Cui, Y. et al. Astroglial Kir4.1 in the lateral habenula drives neuronal bursts in depression. Nature https://doi.org/10.1038/nature25752 (2018)

    Article  ADS  CAS  PubMed  Google Scholar 

  35. 35

    Henn, F. A. & Vollmayr, B. Stress models of depression: forming genetically vulnerable strains. Neurosci. Biobehav. Rev. 29, 799–804 (2005)

    Article  PubMed  PubMed Central  Google Scholar 

  36. 36

    Gigliucci, V. et al. Ketamine elicits sustained antidepressant-like activity via a serotonin-dependent mechanism. Psychopharmacology (Berl.) 228, 157–166 (2013)

    Article  CAS  Google Scholar 

  37. 37

    Gören, M. Z. & Onat, F. Ethosuximide: from bench to bedside. CNS Drug Rev. 13, 224–239 (2007)

    Article  PubMed  PubMed Central  Google Scholar 

  38. 38

    Gideons, E. S., Kavalali, E. T. & Monteggia, L. M. Mechanisms underlying differential effectiveness of memantine and ketamine in rapid antidepressant responses. Proc. Natl Acad. Sci. USA 111, 8649–8654 (2014)

    Article  ADS  CAS  PubMed  Google Scholar 

  39. 39

    Ambert, N. et al. Computational studies of NMDA receptors: differential effects of neuronal activity on efficacy of competitive and non-competitive antagonists. Open Access Bioinformatics 2, 113–125 (2010)

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40

    Endo, M ., Kurachi, Y . & Mishina, M. Pharmacology of Ionic Channel Function: Activators and Inhibitors Vol. 147 (Springer Science & Business Media, 2012)

  41. 41

    Mehrke, G., Zong, X. G., Flockerzi, V. & Hofmann, F. The Ca++-channel blocker Ro 40-5967 blocks differently T-type and L-type Ca++ channels. J. Pharmacol. Exp. Ther. 271, 1483–1488 (1994)

    CAS  PubMed  Google Scholar 

  42. 42

    Hasan, M. et al. Quantitative chiral and achiral determination of ketamine and its metabolites by LC-MS/MS in human serum, urine and fecal samples. J. Pharm. Biomed. Anal. 139, 87–97 (2017)

    Article  CAS  PubMed  Google Scholar 

  43. 43

    Kim, K. S. & Han, P. L. Optimization of chronic stress paradigms using anxiety- and depression-like behavioral parameters. J. Neurosci. Res. 83, 497–507 (2006)

    Article  CAS  PubMed  Google Scholar 

  44. 44

    Powell, T. R., Fernandes, C. & Schalkwyk, L. C. Depression-related behavioral tests. Curr. Protoc. Mouse Biol. 2, 119–127 (2012)

    Article  PubMed  Google Scholar 

  45. 45

    Zhu, Y., Wienecke, C. F., Nachtrab, G. & Chen, X. A thalamic input to the nucleus accumbens mediates opiate dependence. Nature 530, 219–222 (2016)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  46. 46

    Gradinaru, V. et al. Molecular and cellular approaches for diversifying and extending optogenetics. Cell 141, 154–165 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. 47

    Lin, J. Y., Lin, M. Z., Steinbach, P. & Tsien, R. Y. Characterization of engineered channelrhodopsin variants with improved properties and kinetics. Biophys. J. 96, 1803–1814 (2009)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  48. 48

    Ren, M., Cao, V., Ye, Y., Manji, H. K. & Wang, K. H. Arc regulates experience-dependent persistent firing patterns in frontal cortex. J. Neurosci. 34, 6583–6595 (2014)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. 49

    Coulter, D. A., Huguenard, J. R. & Prince, D. A. Calcium currents in rat thalamocortical relay neurones: kinetic properties of the transient, low-threshold current. J. Physiol. (Lond.) 414, 587–604 (1989)

    Article  CAS  PubMed Central  Google Scholar 

  50. 50

    Shabel, S. J., Proulx, C. D., Piriz, J. & Malinow, R. Mood regulation. GABA/glutamate co-release controls habenula output and is modified by antidepressant treatment. Science 345, 1494–1498 (2014)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  51. 51

    Valentinova, K. & Mameli, M. mGluR-LTD at excitatory and inhibitory synapses in the lateral habenula tunes neuronal output. Cell Reports 16, 2298–2307 (2016)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. 52

    Zhu, Z. T., Munhall, A., Shen, K. Z. & Johnson, S. W. Calcium-dependent subthreshold oscillations determine bursting activity induced by N-methyl-d-aspartate in rat subthalamic neurons in vitro. Eur. J. Neurosci. 19, 1296–1304 (2004)

    Article  PubMed  Google Scholar 

  53. 53

    Rateau, Y. & Ropert, N. Expression of a functional hyperpolarization-activated current (Ih) in the mouse nucleus reticularis thalami. J. Neurophysiol. 95, 3073–3085 (2006)

    Article  CAS  PubMed  Google Scholar 

  54. 54

    Bon, C. L., Paulsen, O. & Greenfield, S. A. Association between the low threshold calcium spike and activation of NMDA receptors in guinea-pig substantia nigra pars compacta neurons. Eur. J. Neurosci. 10, 2009–2015 (1998)

    Article  CAS  PubMed  Google Scholar 

  55. 55

    Zhong, P. & Yan, Z. Differential regulation of the excitability of prefrontal cortical fast-spiking interneurons and pyramidal neurons by serotonin and fluoxetine. PLoS One 6, e16970 (2011)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  56. 56

    Caiati, M. D. & Cherubini, E. Fluoxetine impairs GABAergic signaling in hippocampal slices from neonatal rats. Front. Cell. Neurosci. 7, 63 (2013)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. 57

    Lin, L. et al. Large-scale neural ensemble recording in the brains of freely behaving mice. J. Neurosci. Methods 155, 28–38 (2006)

    Article  PubMed  Google Scholar 

  58. 58

    Fries, P., Roelfsema, P. R., Engel, A. K., König, P. & Singer, W. Synchronization of oscillatory responses in visual cortex correlates with perception in interocular rivalry. Proc. Natl Acad. Sci. USA 94, 12699–12704 (1997)

    Article  ADS  CAS  PubMed  Google Scholar 

  59. 59

    Rutishauser, U., Ross, I. B., Mamelak, A. N. & Schuman, E. M. Human memory strength is predicted by theta-frequency phase-locking of single neurons. Nature 464, 903–907 (2010)

    Article  ADS  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank L.-S. Yu and J. Pan for performing LC–MS/MS; S.-M. Duan, X.-H. Zhang, X.-W. Chen, Y.-S. Shu, X. Ju, C. Lohmann and W. Yang for advice on experimental design and comments on the manuscript; and N. Lin, L. Zhang and K.-F. Liu for consultation on in vivo recording. This work was supported by grants from the National Key R&D Program of China (2016YFA0501000), the National Natural Science Foundation of China (91432108, 31225010, and 81527901) to H.H. and (81600954) to Y.Y., and the 111 project (B13026) to H. H.

Author information

Affiliations

Authors

Contributions

H.H, Y.C and Y.Y. designed the study. Y.C. performed the in vitro patch-clamp experiments. Y.Y. and S.M. conducted the behavioural pharmacology experiments. K.S. performed the in vivo recordings. Y.D. and K.S. performed the optogenetic behaviour experiments. Z.N. established the biophysical model. H.H. conceived the project and wrote the manuscript with the assistance of Y.C., Y. Y. and Z.N.

Corresponding author

Correspondence to Hailan Hu.

Ethics declarations

Competing interests

H.H., Y.Y. and Y.C. are inventors on two patent applications (201710322647.X and 201710322646.5) filed on the basis on this work. The remaining authors declare that they have no competing interests.

Additional information

Reviewer Information Nature thanks P. Kenny, H.-S. Shin 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.

Extended data figures and tables

Extended Data Figure 1 Representative ion chromatograms of separation of ketamine by LC–MS/MS and OFT results for different drug treatments.

a, Antidepressant response to systemic ketamine injection (25 mg kg–1, i.p.) in FST 1 h after treatment in cLH rats. n = 10, 8, 6 for wild-type rats injected with saline, cLH rats injected with saline, and cLH rats injected with ketamine, respectively. b, c, OFT of cLH rats after bilateral infusion of ketamine (25 μg, 1 μl each side) or mibefradil (10 nmol, 1 μl each side; b), AP5 (40 nmol, 1 μl each side) or NBQX (1 nmol, 1 μl each side; c) into the LHb. n = 7, 10, 6 rats (b) and 8, 9, 9 rats (c) for saline, ketamine and mibefradil, respectively. d, OFT of CRS mice 1 h after intraperitoneal injection of saline or ethosuximide. n = 6, 9 mice for saline and ethosuximide, respectively. eg, Representative ion chromatograms of separation of ketamine including double blank (e), brain calibrator spiked with 100 ng ml–1 ketamine (KET) (f) and a habenular sample from a cLH rat bilaterally infused with 25 μg μl–1 ketamine into the LHb 1 h earlier (g). Fluvoxamine (FFSM) is used as the internal standard. Data are mean ± s.e.m.; *** P < 0.001, **** P < 0.0001; n.s., not significant. One-way ANOVA (ac), two-tailed Mann–Whitney test and unpaired t-test (d).

Extended Data Figure 2 IV relationship, input resistance and burst duration are not changed in animal models of depression.

ad, IV plots (a), input resistance (b), intra-burst frequency (c) and inter-burst frequency (d) for LHb neurons in brain slices from mice and rats. Note that intra-burst frequency (c) but not inter-burst frequency (d) reversely correlates with RMPs. n = 20 neurons per group, 5 wild-type and 3 cLH rats (b); n = 53 neurons, 8 mice and 12 rats (c, d). ei, Burst duration in animal models of depression recorded in brain slices in vitro (eg) and in behaving animals in vivo (h, i). e, Representative trace of a typical burst in an in vitro recording. It consists of a depolarizing wave and a high-frequency train of action potentials. The duration of the line indicated by the black arrow at half-maximum amplitude of the area under the red dash line is defined as the half width of burst duration; the duration between the first and last shoots (intra burst spikes) within one burst is defined as the shoot duration. f, g, Half widths of burst duration (f) and shoot duration (g) do not differ between cLH and wild-type rats. n = 10, 20 neurons, 7 wild-type and 5 cLH rats. h, Representative trace of an LHb neuron (pink shades indicate burst events) recorded in vivo. An enlarged view of a typical burst on the right shows the definition of burst duration, which is the time interval between the first and last spike within the same burst. i, Burst duration of LHb neurons from in vivo recording do not differ between control and CRS mice. n = 35, 33 neurons, 5 control and 5 CRS mice. Data are mean ± s.e.m.; n.s., not significant. Two-tailed unpaired t-test (b, f, g) and Mann–Whitney test (i).

Extended Data Figure 3 Chronic restraint stress induces reliable depression-like phenotypes and increased burst firing, which can be reversed by ketamine.

a, b, CRS induces increased immobility and decreased latency to immobility in the FST (a) and decreases sucrose preference in the SPT (b). n = 8, 14 mice (a) and 7, 8 mice (b) for control and CRS group, respectively. c, CRS increases locomotion in the OFT. n = 8 mice per group. d, e, Ketamine suppresses immobility of CRS mice in the FST (d) and increases sucrose preference (e) in CRS mice. f, Ketamine decreases locomotion of CRS mice in the OFT. n = 8, 12 mice (d, f) and 14, 21 mice (e) for saline and ketamine groups, respectively. gj, Burst firing is significantly increased in CRS mice, and this increase is reversed by ketamine (i.p., 10 mg kg–1). g, Whole-cell patch-clamp recording sites across different subregions of LHb in mice. h, Pie charts illustrating the per cent abundance of the three types of LHb neurons. i, Bar graph illustrating the percentage of burst- and tonic-type spikes in all spikes recorded. j, Histogram of distribution of inter-spike intervals (ISIs). n = 63, 69, 57 neurons, 4 control, saline-treated mice, 3 CRS, saline-treated mice and 5 CRS, ketamine-treated mice. Data are mean ± s.e.m.; *P < 0.05, **P < 0.01, ****P < 0.0001, n.s., not significant. Two-tailed Mann–Whitney test and unpaired t-test (af), Chi-square test (h), Fisher’s exact test (i).

Extended Data Figure 4 Ketamine suppresses LHb bursting activity in vivo.

a, Histology of recording site. Arrowhead indicates the electrical lesion by tetrodes. b, Representative waveform clusters of two isolated LHb units in the respective four channels of a tetrode (left) and principal-component analysis (PCA) clustering display of these two units (right). c, ISIs between consecutive spikes in relation to their positions within the burst (120 bursts from in vitro recording). d, Example recording trace (upper) and spike train (bottom) of an irregular-firing LHb neuron from an in vivo recording. Bursts (blue sticks) are identified by ISI method (see Methods for details): 1, ISI to start burst; 2, ISI to end burst; 3, inter-burst interval. e, Histogram of ISI distribution (bin, 2.5 ms) from in vivo recording. fi, Mean of total and tonic firing rates (f, h), intra- and inter-burst frequencies, and number of spikes per burst (g, i) of neurons recorded in vivo from control mice, CRS mice and CRS mice 1 h before and after ketamine injection (i.p., 10 mg kg–1). n = 35, 33 neurons (f, g) and 18, 18 neurons (h, i), 5 control and 5 CRS mice. j, STAs of neurons recorded in vivo from control mice, CRS mice, and CRS mice after ketamine injection (i.p., 10 mg kg–1). Note that the distance between the neighbouring troughs is around 140 ms (corresponding to 7 Hz) in CRS mice. Data are mean ± s.e.m.; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, n.s., not significant. Two-tailed Mann–Whitney test and unpaired t-test (f, g), two-tailed Wilcoxon matched-pairs signed rank test and paired t-test (h, i).

Extended Data Figure 5 Drug effects on NMDAR currents, RMPs and miniature EPSCs of LHb neurons.

a, Example traces showing evoked EPSCs when cells were held at –80 mV. NMDAR EPSCs were isolated by application of picrotoxin (100 μM) and NBQX (10 μM) in Mg2+-free ACSF, and confirmed by AP5 (100 μM) blockade. b, Amplitudes of NMDAR EPSCs under different voltages (EPSCs are recorded under 0 Mg2+, picrotoxin and NBQX). Note that isolated NMDAR EPSCs are completely blocked by AP5. cf, Ketamine (c), AP5 (d), NBQX (e) and mibefradil (f) do not affect RMPs of LHb neurons. n = 10 neurons, 5 rats (c); n = 17 neurons, 6 rats (d); n = 11 neurons, 6 rats (e); n = 10 neurons, 3 rats (f). g, ZD7288 causes a small but significant hyperpolarization of LHb neurons. n = 9 neurons, 4 rats. h, Example mEPSC traces before (black) and after (red) perfusion of ketamine (100 μM, see Methods) measured in a whole-cell configuration (cells were held at –60 mV) from LHb neurons. i, j, Cumulative distribution of mEPSC amplitude (i) or mEPSC inter-events interval and average frequency (j) of neurons before (black) or after (red) ketamine treatment. Each line represents values before or after treatment with ketamine from the same LHb neuron. n = 9 neurons, 2 rats. Data are mean ± s.e.m.; *P < 0.05, n.s., not significant. Two-tailed paired t-test.

Extended Data Figure 6 Voltage dependency of LHb bursts and pharmacological manipulation of hyperpolarization-triggered rebound bursts in LHb.

a, Representative trace of an LHb neuron transformed from bursting to tonic-firing mode with a ramp-like current injection, showing bursting at more hyperpolarized potentials and tonic firing at more depolarized potentials. Spikes in bursting and tonic-firing mode are shown in blue and black, respectively. bd, Correlations of membrane potential with intra-burst frequency (b), burst duration (c) and intra-burst spike number (d) generated by current ramps. n = 104 neurons, 14 rats. e, f, Example traces of a spontaneously tonic-firing neuron transformed to burst-firing mode by hyperpolarization (e), and a spontaneously bursting neuron transformed to tonic-firing mode by depolarization (f). gk, Example traces (left) and statistics (right) showing effects of ketamine (g), AP5 (h), mibefradil (i), ZD7288 (j) and fluoxetine (k) on rebound bursts induced by a transient hyperpolarizing current step. Current injection steps are illustrated under the bottom of the trace. n = 9 neurons, 2 rats (g); n = 12 neurons, 5 rats (h); n = 14 neurons, 2 rats (i); n = 8 neurons, 3 rats (j); n = 6 neurons, 2 rats (k). Data are mean ± s.e.m.; *P < 0.05, ****P < 0.0001, n.s., not significant. Two-tailed paired t-test.

Extended Data Figure 7 T-VSCC currents and RMPs of LHb neurons in animal models of depression.

a, Voltage steps used to isolate T-VSCC currents, starting from a holding potential of −50 mV before being increased to conditioning potential (−100 mV) for 1 s preceding the command steps (5 mV, 0.1 Hz per step increment). b, LHb T-VSCC currents (right column) are obtained by subtraction of recorded traces without (left) mibefradil from those with mibefradil (10 μM, middle). The maximum of isolated T-VSCC current is obtained at −50 mV. c, d, No difference in LHb T-VSCC currents is detected between wild-type and cLH rats (c) or control and CRS mice (d). n = 4 neurons per group from 2 wild-type and 2 cLH rats (c); n = 5 neurons per group from 2 control and 2 C57 mice (d). e, f, Scatter plots showing that neuronal RMPs are more hyperpolarized in cLH rats (e) and CRS mice (f) than in controls. n = 45 neurons per group from 5 wild-type and 4 cLH rats (e); n = 50 neurons per group from 4 control and 3 C57 mice (f). Data are mean ± s.e.m.; ***P < 0.001, ****P < 0.0001. Two-tailed unpaired t-test (e, f).

Extended Data Figure 8 Simulation of LHb neurons incorporating T-VSCCs and NMDAR channels.

a, Scheme of a single compartment model of an LHb neuron (see Methods). b, The contribution of NMDAR current INMDAR and T-VSCC current IT during bursts derived from simulation. c, RMP-dependent firing mode of the LHb model neuron. Spikes in bursting mode are shown in blue. Spikes in tonic and silent firing mode are shown in black. d, The correlation between RMPs and intra-burst frequencies of the LHb model neuron. e, f, Example trace (left) and statistics (right) showing in silico effects of ketamine (set NMDAR conductance gNMDAR = 0, e) or mibefradil (gT-VSCC = 0, f) on spontaneous bursts in the LHb model neuron. The bursting probability was evaluated across ten independent trials with simulated synaptic inputs. Note that in silico knockout of NMDAR or T-VSCC current from the model abolished the bursts, which matched experimental observations (Fig. 3a, d, 4a). g, h, Example trace (left) and statistics (right) showing in silico effects of NBQX (gAMPAR = 0, g) or AMPA (gAMPAR increased from 8 to 15 μS cm−2, h) on spontaneous bursts in the LHb model neuron. n = 10 simulations (eh). i, An example trace summarizing the ionic components and channel mechanisms involved in LHb bursting: hyperpolarization of neurons to membrane potentials negative to −55 mV slowly de-inactivates T-VSCC. IT continues to grow as the de-inactivated T-VSCCs increase, leading to a transient Ca2+ plateau potential. The Ca2+ plateau helps remove the magnesium blockade of NMDARs while T-VSCC inactivates rapidly during the depolarization. After the Ca2+ plateau reaches approximately −45 mV, INMDA dominates the driving force to further depolarize RMP to the threshold for Na spike generation. As RMP falls back to below −55 mV it de-inactivates IT and results in the intrinsic propensity of LHb neurons to generate the next cycle of bursting. Data are mean ± s.e.m.; ****P < 0.0001. Two-tailed paired t-test.

Extended Data Figure 9 AMPA or picrotoxin increases LHb burst frequency.

a, b, Example traces (left) and statistics (right, sampled within 3 min before and 1 min after drug application) showing effects of AMPA (a) or picrotoxin (b) on spontaneous bursts in the LHb. n = 8 neurons, 2 rats (a); n = 6 neurons, 3 rats (b). Data are mean ± s.e.m.; *P < 0.05, ***P < 0.001. Two-tailed paired t-test.

Extended Data Figure 10 Additional behavioural results of eNpHR3.0- or oChIEF-based photostimulation.

a, Representative trace showing that LHb neurons can follow only the first of the five 100-Hz pulsed blue light stimulations in a pulsed 100-Hz protocol in LHb brain slices infected with AAV2/9-oChIEF. Percentage of responsive neurons shown on the right. bd, Pulsed 100-Hz photostimulation of mice expressing oChIEF does not change locomotion in the OFT (b), and does not induce depressive phenotypes in the FST (c) or SPT (d). n = 6, 7 mice (b, c) and 5, 5 mice (d) for oChIEF and eGFP groups, respectively. e, Representative trace showing LHb neurons following a 5-Hz tonic blue light stimulation protocol in LHb brain slices infected with AAV2/9- oChIEF. Percentage of responsive neurons shown on the right. fh, 5-Hz photostimulation of LHb in mice expressing oChIEF does not change locomotion in the OFT (f) and does not induce depressive phenotypes in the FST (g) or SPT (h). n = 7, 6 mice (f); 6, 6 mice (g) and 5, 12 mice (h) for oChIEF and eGFP groups, respectively. i, 20-Hz tonic photostimulation of the LHb in mice expressing oChIEF does not cause RTPA. Note that this result is different from 20-Hz optogenetic stimulation of a presynaptic input from the entopeduncular nucleus into the LHb, which caused aversion11. n = 5, 7 mice for oChIEF and eGFP group. j, k, eNpHR3.0-driven burst does not change speed of movement in RTPA (j) or affect locomotion in the OFT (k). n = 11, 7 mice (j) and 13, 21 mice (k) for NpHR and eGFP group, respectively. Data are mean ± s.e.m.; n.s., not significant. Two-way ANOVA (b, d, f, h, k), two-tailed unpaired t-test (c, g, i) and paired t-test (j).

Supplementary information

Supplementary Information

This file contains the model parameters and a detailed model description. (PDF 193 kb)

Life Sciences Reporting Summary (PDF 72 kb)

Supplementary Table 1

This file contains all the statistical analyses and n numbers. (XLSX 27 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Yang, Y., Cui, Y., Sang, K. et al. Ketamine blocks bursting in the lateral habenula to rapidly relieve depression. Nature 554, 317–322 (2018). https://doi.org/10.1038/nature25509

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing