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Emergence of consciousness from anesthesia through ubiquitin degradation of KCC2 in the ventral posteromedial nucleus of the thalamus

Nature Neuroscience volume 26, pages 751–764 (2023)Cite this article

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Abstract

The emergence of consciousness from anesthesia, once assumed to be a passive process, is now considered as an active and controllable process. In the present study, we show in mice that, when the brain is forced into a minimum responsive state by diverse anesthetics, a rapid downregulation of K+/Cl− cotransporter 2 (KCC2) in the ventral posteromedial nucleus (VPM) serves as a common mechanism by which the brain regains consciousness. Ubiquitin-proteasomal degradation is responsible for KCC2 downregulation, which is driven by ubiquitin ligase Fbxl4. Phosphorylation of KCC2 at Thr1007 promotes interaction between KCC2 and Fbxl4. KCC2 downregulation leads to γ-aminobutyric acid type A receptor-mediated disinhibition, enabling accelerated recovery of VPM neuron excitability and emergence of consciousness from anesthetic inhibition. This pathway to recovery is an active process and occurs independent of anesthetic choice. The present study demonstrates that ubiquitin degradation of KCC2 in the VPM is an important intermediate step en route to emergence of consciousness from anesthesia.

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Fig. 1: Downregulation of expression of KCC2 in the thalamus after propofol anesthesia.
Fig. 2: Knockdown of KCC2 in the VPM of the thalamus weakens anesthesia.
Fig. 3: Decreased activity of KCC2 in the VPM leads to GABA-driven Cl– output and EGABA depolarization shift after anesthetic treatment.
Fig. 4: Inhibition of the ubiquitin-proteasome pathway restores KCC2 expression in the VPM and delays recovery from anesthesia.
Fig. 5: The F-box protein Fbxl4 promotes proteasomal degradation of KCC2.
Fig. 6: Effects of manipulation of Fbxl4 expression in the VPM on level of consciousness.
Fig. 7: Phosphorylation of KCC2 at Thr1007 promotes its interaction with Fbxl4.
Fig. 8: Inhibition of KCC2 phosphorylation at Thr1007 by Thr1007A in the VPM in vivo further decreases the VPM neuron excitability and delays recovery from anesthesia.

Data availability

The data that support the findings of the present study are available within this paper and its Supplementary Information. Source data are provided with this paper.

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Acknowledgements

We thank H. Guo for his thoughtful comments and suggestions and W. Song at the University of Pennsylvania Perelman Medical School for manuscript editing. The work was partly supported by Shenzhen Science and Technology Program (grant no. KQTD20200820113040070), the Foundation of Shenzhen Science and Technology Innovation Committee (grant no. JCYJ20200109141433384), the Natural Science Foundation of China (grant no. NSFC81971062) and the Southern University of Science and Technology (grant no. G02416002, all to X.-J.S.).

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Author notes

  1. These authors contributed equally: Jiang-Jian Hu, Yuexin Liu, Hongyu Yao.

Authors and Affiliations

  1. Department of Medical Neuroscience, School of Medicine, Southern University of Science and Technology, Shenzhen, China

    Jiang-Jian Hu, Yuexin Liu, Hongyu Yao, Boxu Cao, Huabao Liao, Peng Chen & Xue-Jun Song

  2. SUSTech Center for Pain Medicine, Southern University of Science and Technology, Shenzhen, China

    Jiang-Jian Hu, Yuexin Liu, Hongyu Yao, Boxu Cao, Huabao Liao, Ruodi Yang, Peng Chen & Xue-Jun Song

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Contributions

J.-J.H. and X.-J.S. designed the project. J.-J.H. and Y.L. performed all experiments and data analyses except for the brain slice electrophysiology experiments and multiple-unit recordings in vivo, which were performed by H.Y. and B.C. H.L., R.Y. and P.C. participated in the collection of behavioral data. J.-J.H. and X.-J.S. wrote the manuscript.

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Correspondence to Xue-Jun Song.

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Extended data

Extended Data Fig. 1 Evaluation of level of consciousness during LORR and expression of KCC2 and NKCC1 in whole brain tissues in MRS.

(a) Consciousness level rating scale and scoring criteria. That this measurement was applied only to animals during the period of LORR, but not for the awake animals. (b) Examples of custom-made tools used in our newly established measurement. From left to right: cotton filaments for evoking blink reflex, neutral resin (50 μl in an Eppendorf tube) for evaluating olfactory response to unpleasant ordour, and free-fall tube for evaluating response to sounds, a plastic tube (29 cm high and 5 cm in diameter) sealed with A4 print-paper (1 cm thick) at the bottom sitting on the wooden surface. A pre-controlled iron cylinder (1 cm high and 1 cm in diameter) was automatically released from top of the plastic tube. (c–e) Time course of changes of consciousness level induced by pentobarbitone (Pen, c), ketamine (Ket, d), and isoflurane (Iso, e). Unlike Pro, Pen and Ket, Iso as an inhaled anesthetic need to be inhaled incessantly to maintain general anesthesia, thus, the behavior was not measured during Iso treatment (30 min). Consciousness level scale scores during LORR, 0 ≤ score < 7; MRS · 3; and RRR = 7 (also see Fig. 1a). n = 6 mice in (c) and (d). (f) Immunoblots of KCC2 expression in the whole brain tissue taken in MRS at 30-min checking point after anesthetic administration. n = 3 mice in each group. **P = 0.0061 (Pro), **P = 0.0075 (Pen), *P = 0.0121 (Ket), and *P = 0.0375 (Iso). (g) Immunoblots of NKCC1 expression in the whole brain tissue taken in MRS induced by propofol. n = 3 mice in each group. P = 0.262. Unpaired Student’s t-test (two-tailed) (f, g). Mean ± s.e.m. MRS versus NA in the same group, *P < 0.05, **P < 0.01, NS = not significant. Detailed statistics information is provided in Supplementary Table 1.

Source data

Extended Data Fig. 2 Expression of KCC2 in the varying brain regions in MRS induced by the diverse anesthetics.

(a) Immunoblots of KCC2 expression in eight different brain regions including the cortex, thalamus, hypothalamus, septum, hippocampus, striatum, brainstem, and cerebellum in MRS induced by Pro, Pen, Iso, and Ket (left to right). n = 3 mice in each group of Pro, Ket, Iso groups and their corresponding NA; n = 4 mice in group of Pen and its corresponding NA. (b) Analysis and comparison of changes of KCC2 expression in the different brain regions in MRS induced by the diverse anesthetics (data from the corresponding groups in a). n = 3 mice in each group of Pro, Ket, and Iso and 4 mice in Pen. Unpaired Student’s t-test (two-tailed). The P values (Pro, Pen, Ket, Iso) in group of cortex: 0.7901, 0.3085, 0.0358, and 0.021; thalamus: 0.0072, 0.0028, 0.0424, and 0.0451; hypothalamus: 0.0005, 0.0016, 0.0028, and 0.0042; septum: 0.2957, 0.0114, 0.8098, and 0.4869; hippocampus: 0.8521, 0.046, 0.0338, and 0.0482; striatum: 0.0945, 0.02641, 0.0333, and 0.7185; brainstem: 0.0138, 0.1061, 0.0024, and 0.2396; cerebellum: 0.0245, 0.1101, 0.9358, and 0.9971. Mean ± s.e.m. MRS versus NA in the same anesthetic group, *P < 0.05, **P < 0.01, ***P < 0.001. Detailed statistics information is provided in Supplementary Table 1.

Source data

Extended Data Fig. 3 Immunofluorescence mapping of expression of KCC2 in the varying brain regions in MRS.

(a–e) Immunofluorescence of KCC2 expression in the thalamus, hypothalamus, cortex, and hippocampus (a) and in the seven sub-nuclei of the thalamus and hypothalamus including MHb, LHb, paraventricular thalamic nucleus (PV)(b), and the VPM (c), and sub-nuclei of the hypothalamus including lateral hypothalamic area (LH), substantia innominate (SI)(d), and paraventricular hypothalamic nucleus (PVN)(e). (f) Data analysis and summary. Mean ± s.e.m., n = 4 mice in each group. One-way ANOVA with Dunnett’s multiple comparisons. P > 0.9999 (NA versus Pro, cortex), P = 0.309 (NA versus Pen, cortex), P = 0.9946 (NA versus Pro, hippocampus). NS = not significant. ****P < 0.0001 in all the others comparisons as indicated. Detailed statistics information is provided in Supplementary Table 1.

Source data

Extended Data Fig. 4 Measuring GABA-driven Cl– transport via fluorescence lifetime measurements of Cl– probe MQAE.

(a) Diagram illustrating the reversal of KCC2 transport due to raising [K+]o (K+-driven Cl– influx). (b) Alterations of efficacy of Cl– transport in the VPM following the diverse anesthetics. Left: representative two-photon lifetime images loaded with Cl– indicator MQAE. Lower lifetime values correspond to higher [Cl–]i. Cell soma are indicated by the circles. Right: data analysis and summary (bin = 10 sec). Anesthetic concentration and number of cells in each group: Pro, 1 mM (n = 34); Pen, 1 mM (n = 34); Iso: 0.11 mM (n = 46); Ket: 0.2 mM (n = 46); vehicle (n = 26); and KCl, 15 mM. Two-way (time × treatment) ANOVA (Mixed-effects model) with Sidak’s multiple comparisons. The P values (each of the anesthetics versus NA): Pro, ****P < 0.0001; Pen, ****P < 0.0001, Ket, ****P < 0.0001, and Iso, **P = 0.0026. Detailed statistics information is provided in Supplementary Table 1.

Source data

Extended Data Fig. 5 Effects of knockdown of KCC2 in the sub-nuclei of the thalamus and hypothalamus on the effect of propofol and isoflurane anesthesia.

(a–f) Representative images showing AAV2/9-hSyn-mCherry-miRNAi (KCC2) transfected LHb, MHb, and PV of the thalamus and LH, PVN, and SI of the hypothalamic nuclei (left two columns). AAV2/9- hSyn-mCherry-miRNAi (KCC2)-induced KCC2 knockdown in these nuclei did not affect propofol (middle two columns) and isoflurane (rightmost column) anesthesia. Number of mice in each of the groups (from a to f): propofol, n = 5, 6, 5, 6, 6, and 6; isoflurane, n = 5, 6, 4, 6, 6, and 6). Unpaired Student’s t-test (two-tailed). The P values (Pro, Iso) in each group: a, P = 0.6454, 0.963; b, P = 0.733, 0.9746; c, P > 0.9999, = 0.9538; d, P = 0.7423, 0.9286; e, P = 0.7294, 0.1372; and f, P = 0.9365. Mean ± s.e.m. NS = not significant. Detailed statistics information is provided in Supplementary Table 1.

Source data

Extended Data Fig. 6 Correlation analysis of curve slope of entering MRS, duration of MRS, and curve slope of exiting from MRS during LORR, related to Fig. 1.

(a) Diagram illustrating the three parameters of curve slope of entering MRS, duration of MRS, and exiting from MRS during LORR. These three curves were firstly fitted linearly and then analyzed for their correlations. (b) Correlation analysis showing no correlation between any two of the three parameters of curve slope of entering MRS, duration of MRS, and exiting from MRS, related to Fig.1b. n = 5 mice. (c–e) Knockdown of KCC2 in VPM did not affect the correlation between any two of the three parameters, related to Fig. 2c, e, g. Number of mice (c-e): n = 6 in each group of miRNAi (NC); n = 7, 6, and 7 in each group of miRNAi(KCC2). Linear regression analysis was performed for any two of the three parameters, and the correlation between the two parameters was analyzed by nonparametric Spearman correlation analysis. P-values (two-tailed) are indicated on each graph, and less than 0.05 was defined as a significant difference. Detailed statistics information is provided in Supplementary Table 1.

Source data

Extended Data Fig. 7 BDNF expression in thalamus and effects of Fblx4 overexpression in the VPM on KCC2 expression in mice with or without propofol anesthesia.

(a) Immunoblots of BDNF expression in thalamus in MRS by Pro. Top, representative western blots. Bottom, data analysis and summary. n = 3 mice in each group. Unpaired Student’s t-test (two-tailed): P = 0.372. (b) Representative western blot images showing time course of changes of expression of KCC2 in the VPM before, during, and recovery from propofol anesthesia following overexpression of Fblx4 by injection of LENTAI–EGFP(Fblx4). (c) Data analysis and summary (from b). n = 3 mice in each group at each checking point. One-way ANOVA with Dunnett’s multiple comparisons and the P values (the specific data point versus NA-uninfected): > 0.9999 (5), < 0.0001 (15), < 0.0001 (30), 0.4151 (90), 0.9998 (120). The P values (the specific data point versus NA-infected): 0.0013 (5), < 0.0001 (15), < 0.0001 (30), 0.0045 (90), 0.9997 (120). Unpaired Student’s t-test (two-tailed). The P values (NA-infected versus NA-uninfected): 0.0041. The P values (infected versus uninfected at the specific data point): 0.0018 (5), 0.0357 (15), 0.3992 (30), 0.0144 (90), and 0.0334 (120). Mean ± s.e.m. ****P < 0.0001, +P < 0.05, ++P < 0.01, ##P < 0.01, ####P < 0.0001. Detailed statistics information is provided in Supplementary Table 1.

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Extended Data Fig. 8 Alterations of expression of phosphorylation of KCC2 Thr1007 in MRS induced by the diverse anesthetics.

(a–c) Immunoblots showing up-regulation of KCC2 Thr1007 phosphorylation in the whole brain (a), thalamus (b), and hypothalamus (c) in MRS with Pro, Pen, Ket, or Iso anesthesia. Phosphorylation of KCC2-Thr1007 increased in the regions where expression of KCC2 decreased (see Extended Data Fig. 2). n = 3 mice in each group of Pro, Ket, Iso and the corresponding NA, n = 4 mice in group of Pen and its corresponding NA. Unpaired Student’s t-test (two-tailed) and the P values (Pro, Pen, Ket, and Iso): a, 0.0146, 0.0377, 0.0293, and 0.0019; b, 0.0006, 0.0122, 0.0401, and 0.012; c, 0.0107, 0.0013, 0.0005, and 0.0437. (d) Immunoblots showing down-regulation of KCC2 Thr1007 phosphorylation in the septum, striatum, cortex, and brainstem. Phosphorylation of KCC2-Thr1007 decreased in the regions where expression of KCC2 increased (see Extended Data Fig. 2). n = 3 mice in each group of Pro, Ket, Iso and the corresponding NA, n = 4 mice in group of Pen and its corresponding NA. Unpaired Student’s t-test (two-tailed) and the P values (Pro, Pen, Ket, and Iso): 0.0154, 0.0088, 0.0003, 0.0322, and 0.002. Mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001. Detailed statistics information is provided in Supplementary Table 1.

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Extended Data Fig. 9 Overexpression of mutant KCC2-Thr1007 (Thr1007A) in the VPM by loci injection of LENTAI–hSyn-EGFP-Thr1007A.

(a) Coronal sections from anterior to posterior confirmed the site of virus injection and infection were well confined to the VPM region. (b) Immunoblots confirmed that overexpression of Thr1007A significantly prevented phosphorylation of KCC2-Thr1007 and degradation of KCC2 in the VPM in MRS with Pen, Ket, or Iso. Left, representative western blots. Right, data analysis and summary. n = 3 mice in each group. One-way ANOVA with Dunnett’s multiple comparisons. The P values (Thr1007/GAPDH): 0.0018 (Pen-Thr1007A versus Pen-NC), 0.0005 (Ket-Thr1007A versus Ket-NC), 0.0001 (Iso-Thr1007A versus Iso-NC). The P values (KCC2/GAPDH): < 0.0001 (Pen-T1007A versus Pen-NC), < 0.0001 (Ket-Thr1007A versus Ket-NC), 0.0055 (Iso-Thr1007A versus Iso-NC). Mean ± s.e.m. **P < 0.01, ***P < 0.001, ****P < 0.00001. Detailed statistics information is provided in Supplementary Table 1.

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Supplementary information

Supplementary Information

Statistical details of tests used in all figures and extended data figures.

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Hu, JJ., Liu, Y., Yao, H. et al. Emergence of consciousness from anesthesia through ubiquitin degradation of KCC2 in the ventral posteromedial nucleus of the thalamus. Nat Neurosci 26, 751–764 (2023). https://doi.org/10.1038/s41593-023-01290-y

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