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. 2013 Dec;19(12):1643-8.
doi: 10.1038/nm.3400. Epub 2013 Nov 17.

Ammonia triggers neuronal disinhibition and seizures by impairing astrocyte potassium buffering

Vinita Rangroo Thrane  1 , Alexander S Thrane, Fushun Wang, Maria L Cotrina, Nathan A Smith, Michael Chen, Qiwu Xu, Ning Kang, Takumi Fujita, Erlend A Nagelhus, Maiken Nedergaard
Affiliations

Ammonia triggers neuronal disinhibition and seizures by impairing astrocyte potassium buffering

Vinita Rangroo Thrane et al. Nat Med. 2013 Dec.

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 death. 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 astrocytes. 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 ammonia. 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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Conflict of interest statement

COMPETING FINANCIAL INTEREST

The authors declare that there are no competing financial interests.

Figures

Figure 1
Figure 1
Ammonia neurotoxicity causes severe neurological impairment and seizures. (a) Diagram showing Otcspf-ash mouse model of acute ammonia neurotoxicity. Ornithine transcarbamylase (Otc), glutamine synthetase (GS), glutamate (Glu), glutamine (Gln), loss of righting reflex (LORR). (b) Automated movement analysis of Otcspf-ash mice given a systemic ammonia load (7.5 mmol kg−1) and saline controls (n = 5–8). (c) Phenotype severity score at different time points after ammonia (n = 8) or saline (n = 5) load in Otcspf-ash mice at time 0. (d) Percent animal freezing on day 3 of spatial fear conditioning comparing Otcspf-ash (n = 5) and wild-type (WT, n = 5) mice. (e) Representative electroencephalogram (EEG) and electromyogram (EMG) recordings of tonic-clonic and myoclonic seizures in ammonia-exposed Otcspf-ash animals (n = 10). (f) Phenotype severity (grey, left axis) compared to myoclonic seizure frequency (red, right axis) with increasing doses of ammonia in Otcspf-ash mice (n = 6 for each). *P < 0.05, **P < 0.01, ***P < 0.001. Data are shown as mean ± SEM.
Figure 2
Figure 2
Ammonia compromises astroglial potassium buffering by competing for uptake. (a) Experimental set-up for studying systemic and cortical ammonia neurotoxicity. 2-photon laser-scanning microscopy (2PLSM), electroencephalogram (EEG). (b) Top, volume analysis of enhanced green fluorescent protein (eGFP) expressing astrocytes during ammonia neurotoxicity (n = 12 for each). Bottom, representative auto-thresholded images (red outline = volume at time 0). Scale bar 5 μm. (c) Left, representative image of eGFP-expressing astrocytes loaded with calcium indicator rhod-2. Scale bar 30 μm, depth 100 μm. Right, corresponding rhod-2 intensity traces (ΔF/F0, eGFP-normalized) before and after a systemic ammonia load (red bar) in Otcspf-ash mouse. (d) Representative NH4+ (top) and K+ (bottom) recordings following systemic ammonia (red bar) or saline (control) load in Otcspf-ash mice. (e) Representative K+ trace (top) and scatterplot of all recordings (bottom, n = 7–10) after cortical application of ammonia (10 mM, red bar). (f) Linear regression of myoclonic seizure frequency on peak cortical [K+]o during systemic and cortical ammonia neurotoxicity (n = 8–10). (g) Left, ouabain-sensitive rubidium (86Rb+) uptake in cultured astrocytes exposed to ammonia, normalized to vehicle (n = 10–23). Right, Na+-K+-ATPase activity measured in 15 mM KCl or NH4Cl solution using a cell-free assay of astrocytes (n = 4 for each). (h) Representative recordings of K+ (top) and myoclonic seizures (bottom) during cortical KCl application (red bar, 12.5 mM, n = 6). Electromyogram (EMG). (i) Diagram of how anatomical (end-feet) and enzymatic (glutamine synthetase, GS) trapping of ammonia in astrocytes compromises their ability to buffer potassium. Glutamine, Gln. Individual myoclonic seizures are indicated by yellow bars throughout. *P < 0.05, **P < 0.01, ***P < 0.001, not significant (NS). Data are shown as mean ± SEM.
Figure 3
Figure 3
Excess ammonia and potassium depolarize the neuronal GABA reversal potential (EGABA) via neuronal Na+-K+-2Cl-cotransporter 1 (NKCC1). (a) Left, diagram of whisker stimulation recordings. Middle, 10 consecutive field excitatory post-synaptic potentials (EPSP) before, during (red) and after cortical ammonia application (10 mM) in wild-type (WT) mice. Right, paired-pulse ratio (PPR) during cortical ammonia application (red bar) and in controls (n = 10 for each). (b) Left, diagram of cortical slice superfused with ammonia (top), and representative ion-sensitive microelectrode (ISM) recordings (bottom). Right, change in ion concentration with 7.5 mM ammonia superfusion (n = 9–18). (c) Representative whole-cell current recordings from pyramidal neurons during ramp voltage, before vs. after GABA ± ammonia application. (d) Current-voltage (I–V) curve comparing effect of ammonia ± bumetanide (BUM) on the currents induced by GABA applications (n = 7–11). (e) Mean EGABA shift caused by ammonia ± bumetanide (n = 7–11). (f) I-V curve of bicuculline and GABA co-application. (g) I-V curve for gramicidin perforated patch recordings before vs. after ammonia exposure in wild-type (left) and Slc12a−/− (right) mice (n = 6–7). (g) Ammonia-induced EGABA shift in wild-type and Slc12a−/− mice (n = 6–7). (h) Immunofluorescence micrographs of Slc12a−/−, Otcspf-ash and wild-type mouse cortex labeled for NKCC1 (green) and nuclei (blue, DAPI). Inset shows choroid plexus. Scale bars 100 μm. (i) Diagram of proposed disease mechanism. Na+-K+-ATPase (NKA), glutamate (Glu), glutamine synthetase (GS), glutamine (Gln), GABAA receptor (GABAAR), K+-Cl− cotransporter isoform 2 (KCC2), membrane potential (Vm). **P < 0.01, ***P < 0.001. Data are shown as mean ± SEM.
Figure 4
Figure 4
Inhibiting Na+-K+-2Cl−cotransporter 1 (NKCC1) with bumetanide treats the electrophysiological and clinical features of ammonia neurotoxicity. (a) Diagram showing bumetanide treatment (BUM, 30 mg kg−1 or 5 μM) for systemic and cortical models of ammonia neurotoxicity. (b) Left, 10 consecutive field recordings during paired-pulse whisker stimulation shown before, during (blue), and after cortical application of ammonia with bumetanide. Right, change in paired-pulse ratio (PPR) following cortical ammonia application (blue box) with bumetanide (n = 10 for each). (c) Automated mouse movement analysis (left) and phenotype severity score (right) after a systemic ammonia load (at time 0) in Otcspf-ash mice with bumetanide treatment (n = 7–8). (d) Effect of bumetanide on myoclonic seizure frequency induced by ammonia and potassium neurotoxicity (n = 6–10). (e) Cox regression of Otcspf-ash mouse survival time after an ammonia overdose (10 mmol kg−1, n = 9–10). *P < 0.05, **P < 0.01, ***P < 0.001. Data are shown as mean ± SEM.
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References

    1. Cagnon L, Braissant O. Hyperammonemia-induced toxicity for the developing central nervous system. Brain Res Rev. 2007;56:183–197. - PubMed
    1. Cooper AJ. 13N as a tracer for studying glutamate metabolism. Neurochem Int. 2011;59:456–464. - PMC - PubMed
    1. Butterworth RF. Pathophysiology of hepatic encephalopathy: a new look at ammonia. Metab Brain Dis. 2002;17:221–227. - PubMed
    1. Jayakumar AR, et al. Na-K-Cl Cotransporter-1 in the mechanism of ammonia-induced astrocyte swelling. J Biol Chem. 2008;283:33874–33882. - PMC - PubMed
    1. Ip YK, Chew SF. Ammonia production, excretion, toxicity, and defense in fish: a review. Front Physiol. 2010;1:134. - PMC - PubMed

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