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Ammonia triggers neuronal disinhibition and seizures by impairing astrocyte potassium buffering

Abstract

Ammonia is a ubiquitous waste product of protein metabolism that can accumulate in numerous metabolic disorders, causing neurological dysfunction ranging from cognitive impairment to tremor, ataxia, seizures, coma and death1. The brain is especially vulnerable to ammonia as it readily crosses the blood-brain barrier in its gaseous form, NH3, and rapidly saturates its principal removal pathway located in astrocytes2. Thus, we wanted to determine how astrocytes contribute to the initial deterioration of neurological functions characteristic of hyperammonemia in vivo. Using a combination of two-photon imaging and electrophysiology in awake head-restrained mice, we show that ammonia rapidly compromises astrocyte potassium buffering, increasing extracellular potassium concentration and overactivating the Na+-K+-2Cl cotransporter isoform 1 (NKCC1) in neurons. The consequent depolarization of the neuronal GABA reversal potential (EGABA) selectively impairs cortical inhibitory networks. Genetic deletion of NKCC1 or inhibition of it with the clinically used diuretic bumetanide potently suppresses ammonia-induced neurological dysfunction. We did not observe astrocyte swelling or brain edema in the acute phase, calling into question current concepts regarding the neurotoxic effects of ammonia3,4. Instead, our findings identify failure of potassium buffering in astrocytes as a crucial mechanism in ammonia neurotoxicity and demonstrate the therapeutic potential of blocking this pathway by inhibiting NKCC1.

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Figure 1: Ammonia causes severe neurological impairment and seizures.
Figure 2: Ammonia compromises astroglial potassium buffering by competing for uptake.
Figure 3: Excess ammonia and potassium depolarize EGABA via NKCC1.
Figure 4: Inhibiting NKCC1 with bumetanide attenuates cortical disinhibition, reduces neurological dysfunction and improves survival following ammonia neurotoxicity.

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Acknowledgements

We thank A.J. Cooper for discussion of the study, S. Kennedy for help with 1H-NMR experiments, L.K. Bekar for help with electrophysiology, J. Chang for designing MatLab software, D. Wang for advice regarding electroencephalogram analysis, J.M. Wilson (University of Pennsylvania) for providing the Otcspf-ash mice, J.D. Rothstein (Johns Hopkins University) for providing Glt1-eGFP BAC transgenic mice and C. Nicholson and S. Hrabetova for advice on fabrication and use of ion-sensitive electrodes. This work was supported by the US National Institutes of Health (grants NS078304 and NS078167 to M.N. and F31NS073390 to N.A.S.), Research Council of Norway (NevroNor FRIMEDBIO grants to E.A.N.), European Commission FP7-ICT-9-601055 to E.A.N., the Molecular Life Science program at the University of Oslo, the Letten Foundation and the Fulbright Foundation.

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Authors

Contributions

V.R.T., A.S.T., E.A.N., M.L.C. and M.N. planned the project. V.R.T., A.S.T., M.L.C., E.A.N. and M.N. wrote the manuscript. V.R.T. and A.S.T. performed in vivo electrophysiology, imaging and data analysis. F.W., N.K. and Q.X. performed in situ electrophysiology. A.S.T., V.R.T. and M.C. performed behavioral experiments. A.S.T., V.R.T. and Q.X. performed in situ imaging. N.A.S. performed rubidium experiments. T.F. performed ATPase experiments. M.L.C. performed immunohistochemistry.

Corresponding author

Correspondence to Alexander S Thrane.

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Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–3 and Supplementary Tables 1 and 2 (PDF 996 kb)

Supplementary Video 1

Myoclonic seizures and decreased spontaneous movement following acute ammonia load in Otcspf-ash mouse. (MP4 7293 kb)

Spontaneous astrocyte calcium signals (baseline).

Two-photon imaging was performed in awake Otcspf-ash mice. Calcium indicator, rhod-2, fluorescence is pseudocolored so that yellow represents higher and blue lower calcium concentration. Time (s) is shown in the top right corner. Scale bar represents 30 μm. (MOV 2945 kb)

Spontaneous astrocyte calcium signals during ammonia neurotoxicity.

Two-photon imaging was performed in awake Otcspf-ash mice. Calcium indicator, rhod-2, fluorescence is pseudocolored so that yellow represents higher and blue lower calcium concentration. Time (s) is shown in the top right corner. Scale bar represents 30 μm. (MOV 1694 kb)

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Rangroo Thrane, V., Thrane, A., Wang, F. et al. Ammonia triggers neuronal disinhibition and seizures by impairing astrocyte potassium buffering. Nat Med 19, 1643–1648 (2013). https://doi.org/10.1038/nm.3400

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