Ammonium chloride alters neuronal excitability and synaptic vesicle release

Abstract

Genetically encoded pH-sensors are widely used in studying cell membrane trafficking and membrane protein turnover because they render exo-/endocytosis-associated pH changes to fluorescent signals. For imaging and analysis purposes, high concentration ammonium chloride is routinely used to alkalize intracellular membrane compartments under the assumption that it does not cause long-term effects on cellular processes being studied like neurotransmission. However, pathological studies about hyperammonemia have shown that ammonium is toxic to brain cells especially astrocytes and neurons. Here, we focus on ammonium’s physiological impacts on neurons including membrane potential, cytosolic Ca2+ and synaptic vesicles. We have found that extracellularly applied ammonium chloride as low as 5 mM causes intracellular Ca2+-increase and a reduction of vesicle release even after washout. The often-used 50 mM ammonium chloride causes more extensive and persistent changes, including membrane depolarization, prolonged elevation of intracellular Ca2+ and diminution of releasable synaptic vesicles. Our findings not only help to bridge the discrepancies in previous studies about synaptic vesicle release using those pH-sensors or other vesicle specific reporters, but also suggest an intriguing relationship between intracellular pH and neurotransmission.

Blinking Fluorescent Probes for Tubulin Nanoscopy in Living and Fixed Cells

“…Importantly, we do not recommend using NH 4 Cl for increasing staining specificity because of the possible disruptive effects on the cell physiology. 23 …”

Long Noncoding RNAs Regulate Hyperammonemia-Induced Neuronal Damage in Hepatic Encephalopathy

“…Hyperammonemia is closely associated with neuronal cell death, the disruption of neuronal morphology, synaptic vesicle release, and long-term synaptic change [ 7 , 31 ]. Patients with minimal hepatic encephalopathy have shown impaired learning and memory, accompanied by changes in the hippocampal structure and function [ 44 ].…”

“…Oxidative Medicine and Cellular Longevity cells exposed to NH 4 Cl metabolize this molecule into ammonia [31]. The SH-SY5Y cells were differentiated into neurons, treated with siRNA against each lncRNA as well as NH 4 Cl, and then used to investigate the role of these lncRNAs during hyperammonemia-induced apoptosis (see Materials and Methods).…”

Fluorescent Measurement of Synaptic Activity Using FM Dyes in Dissociated Hippocampal Cultured Neurons

“…Clearly, both pH manipulations have a possible impact on synaptic vesicle behavior. Notably, our recent paper demonstrated that even a moderate concentration of NH 4 Cl ( i.e ., 5 mM) can change synaptic vesicle release and retrieval over an extended period of time (Lazarenko et al ., 2017). …”

“…The methods were adapted from (Lazarenko et al ., 2017). Techniques were also adapted from all of the references cited.…”