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Combined alcohol and cannabinoid exposure leads to synergistic toxicity by affecting cerebellar Purkinje cells

Nature Metabolism volume 4, pages 1138–1149 (2022)Cite this article

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Abstract

Combined use of cannabis and alcohol results in greater psychoactive toxicity than either substance alone, but the underlying central mechanisms behind this worsened outcome remain unclear. Here we show that the synergistic effect of Δ9-tetrahydrocannabinol (THC) and ethanol on motor incoordination in mice is achieved by activating presynaptic type 1 cannabinoid receptors (CB1R) and potentiating extrasynaptic glycine receptors (GlyR) within cerebellar Purkinje cells (PCs). The combination of ethanol and THC significantly reduces miniature excitatory postsynaptic current frequency in a CB1R-dependent manner, while increasing the extrasynaptic GlyR-mediated chronic chloride current, both leading to decreased PC activity. Ethanol enhances THC actions by boosting the blood–brain-barrier permeability of THC and enriching THC in the cell membrane. Di-desoxy-THC, a designed compound that specifically disrupts THC–GlyR interaction without affecting the basic functions of CB1R and GlyR, is able to restore PC function and motor coordination in mice. Our findings provide potential therapeutic strategies for overcoming the synergistic toxicity caused by combining cannabis and alcohol use.

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Fig. 1: Identification of the role of 4/5Cb neurons in the SETEM.
Fig. 2: Effects of THC and ethanol on PC excitability.
Fig. 3: Pharmacological identification of drug targets in the 4/5Cb that are involved in the SETEM.
Fig. 4: Effects of a CB1R antagonist on the changes in PC neuronal excitability and mEPSCs caused by THC and ethanol combination.
Fig. 5: Effects of THC and ethanol combination on the function of esGlyR.
Fig. 6: Effects of ethanol on THC distribution in HEK293 and N2A cells.
Fig. 7: Effects of ethanol on THC distribution in the brain.
Fig. 8: Effects of DDT on PC activity, the SETEM and other behaviors.

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Acknowledgements

We thank T. Pan for the synthesis of THC, THC–Cy7 and DDT. We thank G.E. Homanics (University of Pittsburgh) for providing the GlyRα1S296A mice. This work was supported by National Key R&D Program of China (2021YFA0804900, W.X.), National Natural Science Foundation of China (grants 91849206, 91649121, 91942315, 92049304, 32121002, 32225020 to W.X.; 81901157 to G.Z.), the Strategic Priority Research Program of the Chinese Academy of Sciences (grant XDB39050000, W.X.), Key Research Program of Frontier Science (CAS, grant ZDBS-LY-SM002, W.X.), CAS Interdisciplinary Innovation Team (JCTD-2018-20, W.X.), CAS Project for Young Scientists in Basic Research (YSBR-013, W.X.), the Fundamental Research Funds for the Central Universities, the Major Program of Development Foundation of Hefei Center for Physical Science and Technology (2017FXZY006, W.X.) and Users with Excellence Program/Project of Hefei Science Center CAS (2019HSC-UE006, W.X.), USTC Research Funds of the Double First-Class Initiative (YD9100002001, W.X.) and China Postdoctoral Science Foundation (2020TQ0314, G.Z.).

Author information

Author notes

  1. These authors contributed equally: Guichang Zou, Jing Xia.

Authors and Affiliations

  1. Institute on Aging and Brain Disorders, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, China

    Guichang Zou, Jing Xia, Heyi Luo, Dan Xiao, Jin Jin, Chenjian Miao, Xin Zuo, Zhi Zhang, Tian Xue & Wei Xiong

  2. Institute of Artificial Intelligence, Hefei Comprehensive National Science Center, Hefei, China

    Guichang Zou & Wei Xiong

  3. CAS Key Laboratory of Soft Matter Chemistry, Department of Polymer Science and Engineering, University of Science and Technology of China, Hefei, China

    Qianqian Gao & Yezi You

  4. Department of Anesthesiology and Perioperative Medicine, The Second Hospital of Anhui Medical University, Hefei, China

    Ye Zhang

  5. Laboratory for Integrative Neuroscience, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, MD, USA

    Li Zhang

  6. Anhui Province Key Laboratory of Biomedical Aging Research, Hefei, China

    Wei Xiong

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  1. Guichang Zou
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Contributions

W.X. initiated, designed and supervised the project; G.Z., C.M. and H.L. conducted electrophysiological recordings. G.Z., J.X. and X.Z. conducted behavior tests; G.Z. and D.X. conducted immunohistochemistry; Y.Y., Q.G. and J.X. conducted the BBB experiments. G.Z. and J.X. conducted the quantitative flow cytometry, in vivo small animal imaging and HPLC–MS experiments. G.Z. and W.X. analyzed data with assistance of T.X, Z.Z. and Y.Z. L.Z. contributed to experimental design, data interpretation and analysis. W.X. and G.Z wrote the manuscript.

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Correspondence to Wei Xiong.

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Extended data

Extended Data Fig. 1 Effects of THC and ethanol on motor coordination and the BEC in mice, Related to Fig. 1.

a, Schematic showing the timeline of drug administration and AR procedure. b, Effects of THC (i.p., 0.1, 0.3, 0.5, 1.0, 3.0 and 5.0 mg/kg) on AR latency of mice. c, Schematic showing the timeline of drug administration and AR procedure. d, Effects of ethanol (i.p., 0.1, 0.3, 0.5, 1.0 and 2.0 g/kg) on AR latency of mice. e, Schematic showing the timeline of drug administration and AR procedure. f, Effects of THC and ethanol in combination on AR latency of mice. g, Effects of ethanol (i.p., 1.0 and 2.0 g/kg) alone or in combination with THC (i.p., 1.0 mg/kg) on AR latency of mice. h, Effects of ethanol on AR latency of female mice. i, Compare of effects of ethanol on AR latency between male and female mice. Each data were normalized to its control group. j, Effects of ethanol (i.p., 0.5, 1.0 and 2.0 g/kg) alone or in combination with THC (i.p., 1.0 mg/kg) on AR latency in female mice. k,l, Normalized AR latency of female (k) and male (l) mice receiving THC (i.p., 1.0 mg/kg) and ethanol (i.p., 0.5, 1.0 and 2.0 g/kg) in combination. Each data were normalized to the related ethanol alone group. m-o, BEC of mice treated with i.p. ethanol at 0.5 g/kg (m), 1.0 g/kg (n) or 2.0 g/kg (o) alone with or without i.p. THC (1.0 mg/kg). n = 3 mice per group. Values are represented as means ± s.e.m., n per group. Exact P values are shown. Statistical differences were determined by a one-way ANOVA followed by Tukey’s post hoc multiple-comparison test (b, d, f-h, j) or a two-way ANOVA followed by Tukey’s post hoc multiple-comparison test (i, k, l, m-o).

Source data

Extended Data Fig. 2 The synergistic effect of THC and various doses of systemic ethanol on c-Fos immunohistochemistry in various brain regions and motor coordination, Related to Fig. 1.

a-c Representative images of immunohistochemistry c-Fos positive neurons in DMS, M1 and M2 of mice treated with Veh, Veh + AR or THC + ethanol + AR. Scale bar, 25 μm. Veh: vehicle, AR: accelerating rotarod. d, Effects of ethanol alone or in combination with THC (intra-4/5Cb., 5 μg/μl) on AR latency of mice. e, Normalized AR latency of mice receiving THC (intra-4/5Cb., 5 μg/μl) and ethanol in combination. Each data were normalized to the ethanol group with the same dose of ethanol. f, Effects of CNO (i.p., 1.0 mg/kg) on the number of c-Fos positive neurons in cerebellar 4/5Cb of mice transfected with AAV2/9-hSyn-hM4Di-mCitrine with or without AR tests. g, Effects of THC (i.p., 1.0 mg/kg) + ethanol (i.p., 0.5 g/kg), CNO (i.p., 1.0 mg/kg), or their combination on the number of c-Fos positive neurons in cerebellar 4/5Cb of mice expressing AAV2/9-hSyn-hM3Dq-mCitrine with or without AR tests. Values are represented as means ± s.e.m., n per group. Exact P values are shown. Statistical differences were determined by a one-way ANOVA followed by Tukey’s post hoc multiple-comparison test (d) or a two-way ANOVA followed by Tukey’s post hoc multiple-comparison test (e-g).

Source data

Extended Data Fig. 3 Effects of chemogenetic activation of 4/5Cb on motor coordination in mice, Related to Fig. 1.

a, Schematic showing the timeline of chemogenetic experimental procedure. AAV2/9-hSyn-hM3Dq-mCitrine or the AAV2/9-hSyn-EGFP was injected into the 4/5Cb of mice. b, Representative images showing the EGFP or mCitrine signals in the injection sites of the 4/5Cb of mice. Scale bar, 1 mm. c, Schematic showing virus injection and chemogenetic experimental procedure. AAV2/9-hSyn-hM3Dq-mCitrine or the AAV2/9-hSyn-EGFP was injected into the 4/5Cb of mice. d, Representative images showing the EGFP or mCitrine signals in the injection sites of the 4/5Cb of mice. Scale bar, 1 mm. e, Effects of CNO (i.p., 1.0 mg/kg) on AR latency of mice expressing hM3Dq or EGFP as a control in the 4/5Cb. Values are represented as means ± s.e.m., n per group. Statistical differences were determined by a two-way ANOVA followed by Tukey’s post hoc multiple-comparison test.

Source data

Extended Data Fig. 4 Effects of THC and ethanol combination on mIPSCs of PCs, Related to Fig. 2.

a, Trace records, average frequency and amplitude of GABAergic mIPSCs of PCs in 4/5Cb slices of mice. The neurons recorded were treated with THC (100 nM, 5 min) and ethanol (20 mM, 5 min), either alone or in combination. b,c, Cumulative probability plot for the inter-event interval (b) and amplitudes (c) for GABAergic mIPSCs of PCs in 4/5Cb slices of mice. Values are represented as means ± s.e.m., n per group. Statistical differences were determined by a one-way ANOVA followed by Tukey’s post hoc multiple-comparison test.

Source data

Extended Data Fig. 5 Effects of various antagonists on SETEM and motor coordination in mice, Related to Fig. 3.

a,b, Effects of AM251 (i.p., 5.0 mg/kg) on the SETEM caused by THC (i.p., 1.0 mg/kg) and ethanol (i.p., 0.5, 1.0 and 2.0 g/kg) combination in male (a) and female (b) mice. c-e, Effects of strychnine (i.p., 1.0 mg/kg) (c), AM630 (i.p., 5.0 mg/kg) (d) and tranilast (i.p., 300 mg/kg) (e) on the SETEM caused by THC (i.p., 1.0 mg/kg) and ethanol (i.p., 1.0 and 2.0 g/kg) combination in mice. Stry: strychnine; Trani: Tranilast. f-i, Effects of AM251(f), strychnine (g), AM630(h) and tranilast (i) on AR latency of mice. j, Effects of AM251 (intra-4/5Cb., 5 μg /μl) on the SETEM caused by THC (i.p., 1.0 mg/kg) and ethanol (i.p., 1.0 and 2.0 g/kg) combination in mice. k, Effects of THC (intra-4/5Cb., 5 μg/μl) and ethanol (i.p., 1.0 g/kg) combination on AR latency in GlyRα1WT and GlyRα1S296A mice. l, Effects of THC (intra-4/5Cb., 5 μg/μl) and ethanol (i.p., 2.0 g/kg) combination on AR latency in GlyRα1WT and GlyRα1S296A mice. m, The AR latency of GlyRα1WT and GlyRα1S296A female mice receiving THC (i.p., 1.0 mg/kg) and ethanol (i.p., 0.5, 1.0 and 2.0 g/kg) combination. Values are represented as means ± s.e.m., n per group. Exact P values are shown. Statistical differences were determined by a one-way ANOVA followed by Tukey’s post hoc multiple-comparison test (a-j) or a two-way ANOVA followed by Tukey’s post hoc multiple-comparison test (k-m).

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Extended Data Fig. 6 Effects of ethanol on Cy7 level in HEK-293 cells and N2A cells measured by standard flow cytometry, Related to Fig. 6.

a-d, Gating strategy to obtain HEK-293 cells with similar size (FSC-A) and granularity (SSC-A). The HEK-293 cells have been incubated with Cy7 (a), Cy7 + ethanol (b), THC-Cy7 (c) and THC-Cy7 + ethanol (d) for 60 min, respectively. Cy7 at 10 μM, THC-Cy7 at 10 μM and ethanol at 20 mM were used in this experiment. FSC-A: Forward Scatter Area; SSC-A: Side Scatter Area. e-h, Gating strategy to obtain N2A cells with similar size (FSC-A) and granularity (SSC-A). The N2A cells have been incubated with Cy7 (e), Cy7 + ethanol (f), THC-Cy7 (g) and THC-Cy7 + ethanol (h) for 60 min, respectively. i,j Histograms (i) and bar graph (j) of MFI (Median Fluorescence Intensity) obtained from standard flow cytometry analysis of HEK-293 cells treated with Cy7 or Cy7 plus ethanol in combination. k,l Histograms (k) and bar graph (l) of MFI obtained from standard flow cytometry analysis of N2A cells treated with Cy7 or Cy7 plus ethanol in combination. m, HPLC calibration curve of HEK-293 cell buffer spiked with known amounts of THC. The calibration curve: y = 2180.40x − 1662.02. n, HPLC calibration curve of N2A cell buffer spiked with known amounts of THC. The calibration curve: y = 2495.71x + 418.599. o,p Representative mass spectra of THC obtained from HEK-293 cell (o) and N2A cell (p) buffer. Values are represented as means ± s.e.m., n per group. Exact P values are shown. Statistical differences were determined by a a two-sided unpaired t-test (j and l) or a linear regression test (m and n).

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Extended Data Fig. 7 Effects of ethanol on Cy7 level in HEK-293 cells and N2A cells measured by imaging flow cytometr, Related to Fig. 6.

a, b Representative histogram used to discriminate between unfocused and focused images of HEK-293 cells (a) and N2A cells (b) incubated with Cy7, Cy7 + ethanol, THC-Cy7 and THC-Cy7 + ethanol. Cy7 at 10 μM, THC-Cy7 at 10 μM and ethanol at 20 mM were used. These experiments were done using the standard IDEAS® software platform. Upon this image, Gradient RMS values between 40–100 (R1: Region 1) were included for further analysis. Gradient RMS: Root Mean Square. c,d Representative graph of area versus aspect ratio used to gating focused images of HEK-293 cells (c) and N2A cells (d). Cells in the frame (R2: Region 2) were used for further membrane intensity analysis. Area: size of the focused cells; Aspect ratio: the minor axis divided by the major axis of the focused cells. e-g, Representative cell images (e), histograms (f) and bar graphs (g) of MFI obtained from IFC (Imaging Flow Cytometry) of HEK-293 cells treated with Cy7 or Cy7 and ethanol in combination. h-j, Representative cell images (h), histograms (i) and bar graphs (j) of MFI obtained from IFC of N2A cells treated with Cy7 or Cy7 and ethanol in combination. Values are represented as means ± s.e.m., n per group. Exact P values are shown. Statistical differences were determined by a two-sided unpaired t-test.

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Extended Data Fig. 8 Effects of various doses of ethanol on plasma and brain intensity of THC and BBB permeability. Related to Fig. 7.

a, Whole body fluorescence imaging of mice treated with Cy7/Cy7-THC (i.p., 10 mg/kg) or Cy7/Cy7-THC (i.p., 10 mg/kg) and ethanol (i.p., 0.5 g/kg) in combination. b, Representative fluorescence images of mice treated with Cy7 alone or Cy7 with ethanol in combination. c, Brain fluorescence intensity of Cy7. n = 5 mice per group. d, Representative fluorescence images of mice treated with THC-Cy7 or THC-Cy7 with ethanol in combination. e, Brain fluorescence intensity of THC-Cy7. n = 5 mice per group. f, Schematic illustration of the in vitro BBB model. g, Relative level of FITC-Dextran (1.0 mg/ml, 1 h) crossing endothelial monolayer with or without ethanol (20 mM, 5 min) treatment. h,i, Representative images (h) and quantitative analysis (i) of FITC signal in the brain of mice treated with FITC-Dextran (i.p., 50 mg/kg, 1 h) with or without ethanol (i.p., 0.5 g/kg, 5 min) administration. Scale bars: 1 mm. j, Calibration curve of plasma spiked with known amounts of THC. The calibration curve: y = 889.307x – 1295.26. k, Representative mass spectra of THC obtained from plasma of C57BL/6 J mice administrated with THC (i.p., 10 mg/kg). l,m Quantitative analysis of plasma (l) and cerebellar (m) THC intensity in mice treated with THC or THC and ethanol in combination. n = 3 mice per group. Values are represented as means ± s.e.m., n per group. Exact P values are shown. Statistical differences were determined by a two-sided unpaired t-test (g and i) or a two-way ANOVA followed by Tukey’s post hoc multiple-comparison test (c, e, l and m) or a linear regression test (j).

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Extended Data Fig. 9 Effects of DDT on acute and chronic ethanol exposure associated SETEM and ethanol-induced motor incoordination, Related to Fig. 8.

a, Schematic illustration of synthesis of di-desoxy-THC (DDT). b, AR latency of mice receiving THC (i.p., 1.0 mg/kg), ethanol (i.p., 1.0 and 2.0 g/kg), DDT (i.p., 30 mg/kg), or their combination. c, Effects of DDT (i.p., 30 mg/kg) on motor incoordination caused by various doses of ethanol. d, Schematic illustration of the CEE. e, The effects of THC and DDT on the CEE-induced motor incoordination. f, The effects of THC and AM251 on the CEE-induced motor incoordination. g, Representative images showing a mouse walking on treadmill and its paw prints detected from a video underneath the treadmill. LF: left forelimb; RF: right forelimb; LH: left hindlimb; RH: right hindlimb. h-j, Effects of DDT (i.p., 30 mg/kg, 20 min before tests) on the stride length (h), stride duration (i) and stance width (j) of mice in the gait test. k-m, Effects of DDT (i.p., 30 mg/kg) on grip strength (k), body temperature (l) and food intake (m) of mice in the grip test. Values are represented as means ± s.e.m., n per group. Exact P values are shown. Statistical differences were determined by a one-way ANOVA followed by Tukey’s post hoc multiple-comparison test (b, c, e and f) or a two-way ANOVA followed by Tukey’s post hoc multiple-comparison test (h-j) or a two-sided unpaired t-tests (k–m).

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Zou, G., Xia, J., Luo, H. et al. Combined alcohol and cannabinoid exposure leads to synergistic toxicity by affecting cerebellar Purkinje cells. Nat Metab 4, 1138–1149 (2022). https://doi.org/10.1038/s42255-022-00633-6

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