Abstract
The mechanisms of volatile anesthetic action remain among the most perplexing mysteries of medicine. Across phylogeny, volatile anesthetics selectively inhibit mitochondrial complex I, and they also depress presynaptic excitatory signaling. To explore how these effects are linked, we studied isoflurane effects on presynaptic vesicle cycling and ATP levels in hippocampal cultured neurons from wild-type and complex I mutant (Ndufs4(KO)) mice. To bypass complex I, we measured isoflurane effects on anesthetic sensitivity in mice expressing NADH dehydrogenase (NDi1). Endocytosis in physiologic concentrations of glucose was delayed by effective behavioral concentrations of isoflurane in both wild-type (τ [unexposed] 44.8 ± 24.2 s; τ [exposed] 116.1 ± 28.1 s; p < 0.01) and Ndufs4(KO) cultures (τ [unexposed] 67.6 ± 16.0 s; τ [exposed] 128.4 ± 42.9 s; p = 0.028). Increasing glucose, to enhance glycolysis and increase ATP production, led to maintenance of both ATP levels and endocytosis (τ [unexposed] 28.0 ± 14.4; τ [exposed] 38.2 ± 5.7; reducing glucose worsened ATP levels and depressed endocytosis (τ [unexposed] 85.4 ± 69.3; τ [exposed] > 1,000; p < 0.001). The block in recycling occurred at the level of reuptake of synaptic vesicles into the presynaptic cell. Expression of NDi1 in wild-type mice caused behavioral resistance to isoflurane for tail clamp response (EC50 Ndi1(−) 1.27% ± 0.14%; Ndi1(+) 1.55% ± 0.13%) and halothane (EC50 Ndi1(−) 1.20% ± 0.11%; Ndi1(+) 1.46% ± 0.10%); expression of NDi1 in neurons improved hippocampal function, alleviated inhibition of presynaptic recycling, and increased ATP levels during isoflurane exposure. The clear alignment of cell culture data to in vivo phenotypes of both isoflurane-sensitive and -resistant mice indicates that inhibition of mitochondrial complex I is a primary mechanism of action of volatile anesthetics.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 3016-3032.e3 |
| Journal | Current Biology |
| Volume | 32 |
| Issue number | 14 |
| DOIs | |
| State | Published - Jul 25 2022 |
Funding
The authors are indebted to the laboratories of Drs. Robert Edwards (UCSF, San Francisco, USA) and Susan Voglmaier (UCSF, San Francisco, USA), who provided significant initial technical assistance and sharing of pHluorin constructs, and to Dr. Navdeep Chandel (Northwestern University, Chicago, IL), who kindly shared the NDi1-LSL mouse and guided us in its use. We also thank Dr. Hiromi Imamura (Kyoto University, Japan) for sharing the FRET constructs. In addition, we thank Drs. Hugh Hemmings (Weill Cornell Medical College, NYC, USA) and Kira Spencer for their open and insightful discussions. Finally, the authors thank Dr. Beatrice Predoi for her continuous excellent technical assistance. P.G.M. M.M.S. J.-M.R. P.I.Z. and S.J. were each supported in part by NIH grants R01 GM122899 and R35GM139566. K.N. and D.H. were each supported in part by NIH Grant R01 NS091902. C.R.R. was supported in part by NIH Grant NHLBI T32 HL076139-11. S.J.: conceptualization, investigation, formal analysis, writing – review & editing. P.I.Z.: conceptualization, investigation, software, formal analysis, writing – review & editing. E.-B.K.: investigation, formal analysis, writing – review & editing. C.B.W.: conceptualization, investigation, writing – review & editing. D.H.: conceptualization, writing – review & editing. C.R.R.: methodology, resources, writing – review & editing. K.N.: conceptualization, formal analysis, writing – review & editing. J.-M.R.: conceptualization, validation, formal analysis, writing – review & editing. M.M.S.: supervision, conceptualization, validation, formal analysis, writing – original draft, and funding acquisition. P.G.M.: supervision, conceptualization, validation, investigation, formal analysis, writing – original draft, and funding acquisition. The authors declare no competing interests. We worked to ensure sex balance in the selection of non-human subjects. The authors are indebted to the laboratories of Drs. Robert Edwards (UCSF, San Francisco, USA) and Susan Voglmaier (UCSF, San Francisco, USA), who provided significant initial technical assistance and sharing of pHluorin constructs, and to Dr. Navdeep Chandel (Northwestern University, Chicago, IL), who kindly shared the NDi1-LSL mouse and guided us in its use. We also thank Dr. Hiromi Imamura (Kyoto University, Japan) for sharing the FRET constructs. In addition, we thank Drs. Hugh Hemmings (Weill Cornell Medical College, NYC, USA) and Kira Spencer for their open and insightful discussions. Finally, the authors thank Dr. Beatrice Predoi for her continuous excellent technical assistance. P.G.M., M.M.S., J.-M.R., P.I.Z., and S.J. were each supported in part by NIH grants R01 GM122899 and R35GM139566 . K.N. and D.H. were each supported in part by NIH Grant R01 NS091902 . C.R.R. was supported in part by NIH Grant NHLBI T32 HL076139-11 .
Keywords
- ATP
- complex I
- endocytosis
- exocytosis
- mitochondria
- mouse
- presynapse
- volatile anesthetics
ASJC Scopus subject areas
- General Biochemistry, Genetics and Molecular Biology
- General Agricultural and Biological Sciences