Astroglial Kir4.1 in the lateral habenula drives neuronal bursts in depression


Enhanced bursting activity of neurons in the lateral habenula (LHb) is essential in driving depression-like behaviours, but the cause of this increase has been unknown. Here, using a high-throughput quantitative proteomic screen, we show that an astroglial potassium channel (Kir4.1) is upregulated in the LHb in rat models of depression. Kir4.1 in the LHb shows a distinct pattern of expression on astrocytic membrane processes that wrap tightly around the neuronal soma. Electrophysiology and modelling data show that the level of Kir4.1 on astrocytes tightly regulates the degree of membrane hyperpolarization and the amount of bursting activity of LHb neurons. Astrocyte-specific gain and loss of Kir4.1 in the LHb bidirectionally regulates neuronal bursting and depression-like symptoms. Together, these results show that a glia–neuron interaction at the perisomatic space of LHb is involved in setting the neuronal firing mode in models of a major psychiatric disease. Kir4.1 in the LHb might have potential as a target for treating clinical depression.

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Figure 1: Kir4.1 is upregulated in the LHb in rat models of depression.
Figure 2: Kir4.1 is expressed on astrocytic processes that wrap tightly around neuronal somata in LHb.
Figure 3: Astrocytic kir4.1 overexpression increases neuronal bursts in the LHb and causes depression-like phenotypes.
Figure 4: Kir4.1-dependent potassium buffering regulates neuronal RMP and bursting in LHb.
Figure 5: Loss of function of Kir4.1 in LHb decreases neuronal bursting and rescues depression-like phenotypes of cLH rats.


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We thank K. McCarthy for Kir4.1 floxed mice; B. Khakh for the GFAP-Kir4.1 plasmid; T. Xue for advice on dn-Kir4.1 design; Y.-Y. Liu for technical support on electromicroscopy; C. Liu and C.-J. Shen for help with immunohistochemistry; S.-M. Duan, Y.-D. Zhou, J.-W. Zhao, X.-H. Zhang and B. MacVicar for advice on experimental design; and C. Giaum and P. Magistretti for comments on the manuscript. 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., 81701335 to Y.C., and 81730035 to S.W.), the Strategic Priority Research Program (B) of the Chinese Academy of Sciences (XDB02030004) and 111 project (B13026) to H.H.

Author information




Y.C. performed the in vitro patch recordings; Y.Y. performed the biochemistry and immunohistochemistry experiments; Y.C., Y.Y., Y.D. and K.S. performed viral injections and behavioural experiments; Z.N., A.F. and H.B. established the biophysical model; S.M. assisted with cell culture experiments; G.C. and S.W. conducted the electron microscopy experiments; Y.S. contributed Kir4.1 floxed mice; S.T. and Y.L. constructed plasmids; H.H. and Y.C. designed the study; and H.H. wrote the manuscript with the assistance of Y.C., Y.Y. and Z.N.

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Correspondence to Hailan Hu.

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Reviewer Information Nature thanks P. Kenny and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Figure 1 Habenular protein expression in rat models of depression.

a, Volcano plot of high-throughput proteomic screen identifies proteins that are differentially expressed in the habenulae of cLH rats versus wild-type rats. Ln (fold change) is ln-transformed value of the normalized protein ratio of cLH and control14. Significance value was calculated as the average normalized ratio minus two folds of s.d.14. Proteins in the shaded areas have more than 50% significant change. Kir4.1 is one of the eight upregulated proteins identified14. Dashed lines indicate fold change of 50%. b, Western blot analysis showing no change in GFAP protein in habenulae of cLH rats at P60–90. n = 4, 4 rats for control and cLH, respectively. c, LPS injection (500 μg kg−1 i.p. for 7 days) induces increased immobile time and decreased latency to immobility in the FST. n = 8, 9 rats for saline and LPS, respectively. d, QPCR analysis of Kir4.1 mRNA in habenulae. Two-tailed paired t-test. n = 5, 5 rats for control and cLH, respectively. e, Western blot analysis showing no change in Kir4.1 protein in membrane fraction of habenulae in cLH rats at P30. n = 6, 6. Data are means ± s.e.m., *P < 0.05, n.s., not significant. Two-tailed paired t-test (b, d, e); two-tailed unpaired t-test (c).

Extended Data Figure 2 Ba2+-sensitive Kir4.1 current is upregulated in LHb of adult cLH rats and adult LPS-injected Wistar rats.

a, c, Representative traces showing linear IV curve in a typical astrocyte before (upper) and after (middle) Ba2+ perfusion under voltage steps (–130 mV to –30 mV, step by 10 mV, 2 s duration, holding at –70 mV). Subtraction of the two led to Ba2+-sensitive Kir current (bottom) at P60–90 (a) and P30 (c) in cLH rats. b, d, IV plots of astrocytes in cLH rats and controls at P60–90 (b) and P30 (d). e, IV plots of astrocytes in LPS-injected Wistar rats and saline controls at P60–90. f, IV plot and bar graph showing Ba2+-sensitive currents in LPS-injected Wistar rats and saline-injected controls at P60–90. Two-tailed unpaired t-test. n = 7, 6 astrocytes from 2, 2 rats for saline and LPS, respectively. Data are means ± s.e.m., *P < 0.05.

Extended Data Figure 3 Biocytin intercellular filling and double staining with NeuN confirm the identity of eletrophysiologically identified neurons and astrocytes.

a, b, A neuron (a) and an astrocyte (b) in LHb slices were first identified on the basis of their specific morphology (astrocytes: 5–10 μm diameter; neurons: ~15 μm diameter) and physiological properties. The neuron fires at a depolarizing voltage step (a), whereas the astrocyte shows a steady-state IV relationship and a lack of spiking activity (b). After electrophysiological characterization, cells were held for at least 30 min in voltage clamp and constantly injected with a hyperpolarization current (500 ms, 50 pA, 0.5 Hz, 30 min) to allow biocytin filling. ch, Biocytin-labelled neurons and astrocytes subsequently confirmed by co-labelling with NeuN. c, d, Biocytin signals in a single neuron (c) or a group of astrocytes owing to diffusion through gap junctions (d) (four independent experiments). e, f, NeuN signals (four independent experiments). g, h, Colabelling of NeuN with the neuron (indicated by white arrow, g) but not astrocytes (h) (four independent experiments). Note that all biocytin-filled neurons (n = 18) colabel with NeuN and all biocytin-filled astrocytes (n = 11) do not colabel with NeuN.

Extended Data Figure 4 Expression pattern of Kir4.1 in the LHb and hippocampus.

a, b, Kir4.1 co-immunostaining with neuronal marker (NeuN) or astrocytic marker (S100b and GFAP) in the LHb (a) or hippocampus (b). Bottom two panels show staining with the same Kir4.1 antibody pre-incubated with the antigen peptide, demonstrating the specificity of the Kir4.1 antibody (two independent experiments).

Extended Data Figure 5 Electron microscopy immunohistochemistry of Kir4.1 staining.

a, b, Many Kir4.1 immunograins (arrows) surround the neuronal soma. c, Kir4.1 grains (arrows) also surround axon–dendrite synapses, but are rare near the synaptic zones as indicated by the postsynaptic densities (arrowheads). d, Kir4.1 immunograins are also detected surrounding a vascular endothelial cell. e, Inset shows Kir4.1 immunograins near a gap junction. s, neuronal soma; t, axon terminal. Scale bars, 0.5 μm. Three independent experiments.

Extended Data Figure 6 Kir4.1 is expressed in astrocytes but not neurons in the LHb.

a, Schematics showing sequence of drug application and recording after a neuron or astrocyte is patched. b, Representative traces showing a linear IV curve in a typical astrocyte under voltage steps (−130 mV to −30 mV, step by 10 mV, 2 s duration, holding at −70 mV, protocol demonstrated on left, upper panel). IV curves of the same cell after addition of TTX (1 μM), 4-aminopyridine (4AP, 1 mM) and 4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino) pyrimidinium chloride (ZD7288, 50 μM) (middle) and further addition of Ba2+ (100 μM, bottom) are shown below. c, Representative traces showing a nonlinear IV curve in a typical neuron under voltage steps (−120 mV to −40 mV, step by 10 mV, 2 s duration, holding at −60 mV, protocol demonstrated on left, upper panel). IV curves of the same cell after addition of TTX, ZD7288 and 4AP (middle) and further addition of Ba2+ (bottom) are shown below.

Extended Data Figure 7 Characterization of cell-type specificity of GFAP promoter, and locomotion.

a, b, Double immunofluorescence for NeuN (red) and eGFP (green) in the coronal section of LHb brain slices infected with AAV-GFAP::Kir4.1 (AAV2/5-gfaABC1D-eGFP-Kir4.1) virus in mice (three independent experiments, a) or AAV-GFAP::dnKir4.1 (AAV2/5-gfaABC1D-dnKir4.1-2A-eGFP) virus in cLH rats (two independent experiments, b). Left, examples of anterior, middle and posterior coronal sections of LHb. Numbers in the bottom right corner are the number of merged cells/number of NeuN+ cells in the virus-infected area. Right, zoomed-in images of the white square area in left. Note that there is only one infected neuron, as indicated by the white arrow, in all three fields of view. c, d, Overexpression of Kir4.1 in the LHb of C57 mice does not affect locomotion. n = 7, 8 mice for eGFP and Kir4.1, respectively. e, f, Overexpression of Kir4.1-shRNA in the LHb of cLH rats does not affect locomotion activities. n = 7, 7 rats for control and Kir4.1 shRNA, respectively. Data are means ± s.e.m; n.s., not significant. Two-tailed unpaired t-test (cf).

Extended Data Figure 8 Simulation of the dynamic potassium buffering effect of Kir4.1 in the tri-compartment model.

a, Schematic representation of a tri-compartment model involving neuron, astrocyte and extracellular space (see Methods for details). bd, Effects of increasing Kir4.1 expression on [K]out (b), neuronal membrane potential (c) and astrocytic membrane potential (d). Ctrl, control condition with 1 × Kir4.1 conductance; Depr, depression condition with 2 × Kir4.1 conductance. Grey shaded areas indicate application of 10 Hz tonic stimulation to neurons. Note that under this neuronal firing condition, [K]out is lower, and neuron and astrocyte are more hyperpolarized in the depression condition than the control. eg, Effects of in silico TTX (blocking action potentials, gNa = 0) or Ba2+ (blocking Kir4.1, gKir4.1 = 0) treatments on [K]out (e), neuronal membrane potential (f) and astrocytic membrane potential (g) when neurons are under 10 Hz tonic stimulation. Grey shaded areas indicate in silico application of drugs. Note that TTX and Ba2+ cause opposite changes to [K]out, neuronal membrane potential and astrocytic membrane potential. Neuronal spikes are not shown for clarity of presentation.

Extended Data Figure 9 BaCl2 caused depolarization of neuronal RMP in the presence of synaptic transmitter blockers.

a, b, Representative trace (a) and bar graph (n = 9 neurons from 3 rats; b) showing effect of BaCl2 (100 μM) perfusion onto tonic-firing neurons that have been bathed with transmitter blockers (100 μM picrotoxin, 10 μM NBQX and 100 μM AP5). c, Bar graph showing the level of RMP depolarization caused by BaCl2 in the presence or absence of transmitter blockers. n = 9, 12 neurons from 3, 3 rats for with and without blockers, respectively. d, Representative trace showing effect of BaCl2 (sampled 15 min after drug perfusion) on bursting neurons (n = 4 out of 9 neurons from 3 rats). Spikes in bursting and tonic-firing mode are shown in blue and black, respectively. Data are means ± s.e.m., ***P < 0.001, n.s., not significant. Two-tailed paired t-test (b) and two-tailed unpaired t-test (c).

Extended Data Figure 10 Characterization of Kir4.1 loss-of-function constructs.

a, Flag-tagged-Kir4.1 plasmid (pAAV-CMV-betaGlobin-Kir4.1-eGFP-3Flag) was co-transfected with pAAV-vector expressing six different shRNAs (see Methods) of Kir4.1 or the negative control (shRNA of luciferase) into HEK293 TN cells. On the basis of knockdown efficiency as shown in the western blot, Kir4.1-shRNA-5 was chosen for viral package (two independent experiments). b, IV plot showing Kir4.1 currents recorded in HEK293 cells transfected with pAAV-Kir4.1 together with negative control pAAV-eGFP or pAAV-dnKir4.1 plasmid. Bars represent the current values recorded at –160 mV. n = 18, 15 HEK293 cells for eGFP and dnKir4.1, respectively. c, IV plot and bar graph showing Ba2+-sensitive currents blocked by AAV-dnKir4.1 in both cLH and wild-type rats. df, AAV-dnKir4.1 caused depolarization of RMP in astrocytes (n = 9, 8, 9, 6 astrocytes from 2, 4, 2, 4 rats for wild-type eGFP, wild-type dnKir4.1, cLH eGFP and cLH dnKir4.1, respectively; d) and neurons in viral infected area (n = 54, 48, 45, 58 neurons from 2, 4, 2, 4 rats, e), and abolished neuronal bursting (f) in both cLH and wild-type rats. Data are means ± s.e.m.; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Two-tailed unpaired t-test (be) and χ2 test (f).

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Cui, Y., Yang, Y., Ni, Z. et al. Astroglial Kir4.1 in the lateral habenula drives neuronal bursts in depression. Nature 554, 323–327 (2018).

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