TY - JOUR T1 - Glutamate, AP5 and NMDA do not directly modulate glycine receptors JF - Molecular Pharmacology JO - Mol Pharmacol DO - 10.1124/molpharm.120.000127 SP - MOLPHARM-AR-2020-000127 AU - Karin R. Aubrey AU - Diba Sheipouri AU - Thomas Balle AU - Robert J. Vandenberg AU - Yo Otsu Y1 - 2020/01/01 UR - http://molpharm.aspetjournals.org/content/early/2020/10/13/molpharm.120.000127.abstract N2 - Reproducibility of research data is a substantial problem with more than 60% of biological and medical researchers reporting they have failed to reproduce published data. The general acceptance of incorrect results can mean that future data is incorrectly interpreted and progress significantly interrupted. Thus, replication studies play an essential role in corroborating research findings and validating future research objectives. Here, we attempted to replicate data demonstrating the neurotransmitter glutamate, as well as NMDA and AP5, directly act at inhibitory glycine receptors as positive allosteric modulators. Consequently, the amplitude of miniature glycinergic currents in spinal slices increased when the glutamate transporter inhibitor TBOA inhibited synaptic glutamate uptake. This suggests that endogenous fluctuations in extracellular glutamate concentrations permit rapid crosstalk between excitatory and inhibitory synapses and likely plays a role in setting the spinal inhibitory glycinergic tone and modulating baseline neurotransmission. We re-evaluated the data in primary cultured spinal cord neurons, spinal cord slice, and Xenopus laevis oocytes expressing recombinant human glycine receptors. Despite extensive efforts, we were unable to reproduce the finding that glutamate, AP5 and NMDA positively modulate glycine receptor currents. We paid careful attention to critical aspects of the original study design and took into account receptor saturation and protocol deviations such as animal species. Finally, we explored possible explanations for the experimental discrepancy. We found that solution contamination with a high-affinity modulator such as zinc is most likely to account for the error and suggest methods for preventing this kind of misinterpretation in future studies. Significance Statement A previous study indicated that glutamate spills-over onto inhibitory synapses can directly interact with glycine receptors to enhance inhibitory signalling. This finding has important implications for baseline spinal transmission and may contribute to chronic pain conditions. However, we failed to replicate the results and did not observe glutamate, AP5 or NMDA modulation of native or recombinant glycine receptors. We ruled out various sources for the discrepancy and found that the most likely cause of the error is solution contamination. ER -