Ibed in cell culture and kidney4,6,17,18. Making use of NH4+ and K+ ionsensitive microelectrodes (ISM)19 in awake Otcspf-ash animals we found that systemic ammonia intoxication improved extracellular potassium ([K+]o) by 1.93 0.19 mM (subtracted for interference17, Fig. 2d). As previous studies and our results indicate that the bulk of ammonia neurotoxicity occurs in cortical grey matter (Supplementary Fig. 2a)two,20, we next applied ammonia straight around the cortex of awake wild-type mice. The ammonia dose was titrated in initial experiments to attain a [NH4+]o improve of 5.82 0.18 mM, which was comparable to our observations from systemic toxicity and adequate to reproduce the seizure phenotype (Supplementary Table 1). We also identified that cortical ammonia intoxication in wild-type mice elevated [K+]o by 2.24 0.17 mM from a resting level of 3.91 0.27 mM (pooled control data) (Fig. 2e, Supplementary Fig. 2b ). The [K+]o raise in both the systemic and cortical models were strongly correlated with and regularly preceded myoclonic seizures (systemic four.91 0.35 min and cortical 2.88 0.20 min), and recovered on washout as the neurological manifestations subsided (Fig. 2f). Conversely, the pH effects of ammonia were mild, delayed and correlated poorly with clinical phenotype (Supplementary Fig. 2e). Having established that ammonia increases [K+]o sufficiently to trigger neurological dysfunction in vivo, we proceeded to ask: Does the excess NH4+ load on astrocyte membranes impair potassium buffering by inhibiting transport or straight competing for uptake The gradients driving astrocyte uptake of potassium are largely dependent on Na+K+-ATPase (NKA) activity, in particular inside the context of sustained [K+]o elevations17,21,22.Ziltivekimab Working with biologically relevant concentrations of ammonia (0.50 mM), we demonstrated a dose-dependent reduction in NKA-mediated (ouabain-sensitive) potassium analogue rubidium (86Rb+) uptake in cultured astrocytes (Fig. 2g, Supplementary Fig. 2f)21. We then substituted KCl with NH4Cl in an astroglial NKA assay, and located that NH4Cl alone was able to keep typical NKA activity (Fig. 2g), indicating that NH4+ competes with K+ forAuthor Manuscript Author Manuscript Author Manuscript Author ManuscriptNat Med.Sutimlimab Author manuscript; out there in PMC 2014 June 01.Thrane et al.Pagetransport. Notably, NH4+ and K+ ions have a comparable hydrated radius and charge6. To test regardless of whether the [K+]o increase alone is enough to reproduce the phenotype of ammonia neurotoxicity in vivo, we superfused KCl (12.PMID:23509865 5 mM) across the cortex of awake wild-type mice. The KCl superfusion improved [K+]o by two.37 0.03 mM and triggered the mice to create myoclonic seizures equivalent to those seen in ammonia neurotoxicity (Fig. 2h). Combined, our observations indicate that ammonia short-circuits potassium transport in astrocytes, and that the consequent boost in [K+]o alone is adequate to bring about neurological dysfunction. Because astrocyte calcium transients potently stimulate NKA activity21, NKA activity is identified to increase in hyperammonemia13, and increased NKA pumping causes cell shrinkage (3Na+ export vs. 2K+ import), we speculate that our imaging observations represent compensatory adjustments in astrocytes to buffer the excess substrate ([NH4+]o and [K+]o) (Fig. 2i). How does impaired astrocyte potassium buffering lead to neurological dysfunction and seizures Preceding ex vivo research have generated many conflicting hypotheses in regards to the impact of ammonia on neur.