Otsu, Y. | Search bioRxiv

bioRxiv2020

Glycine receptors are not directly modulated by glutamate, AP5 or NMDA

3.Reproducibility of research data is a significant problem with more than 60% of biological and medical researchers reporting they have failed to reproduce published data. 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, acts as positive allosteric modulators of the inhibitory glycine receptor. Notably, it was shown that the amplitude of miniature glycinergic currents recorded in spinal cord slices were reversibly enhanced when extracellular glutamate concentrations were increased by the glutamate transporter antagonist TBOA. This finding indicates that endogenous fluctuations in extracellular [glutamate] permits cross-talk 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 glycine receptors. Despite extensive efforts, we were unable to reproduce the finding that glutamate, AP5 or NMDA positively modulate glycine receptor currents. We paid careful attention to key aspects of the original study design, ensured rapid drug exposure by using fast-flow application and took into account receptor saturation and protocol deviations such as animal species. This study refutes the finding that glycine receptors are directly modulated by glutamate spill-over and suggests that glycinergic tone is independent of changes in excitatory activity. 4. Significance StatementGlutamate spill-over onto inhibitory synapses has been reported to positively modulate glycine receptors and alter the inhibitory tone of the spinal cord. This finding has important implications for baseline spinal transmission and could play a role when chronic pain develops. However, we failed to replicate these results and did not observe any modulation of native or recombinant glycine receptor-mediated currents by AP5, NMDA or glutamate. This indicates that inhibitory glycine receptors operate independently of fluctuations in extracellular [glutamate]. 5. Visual AbstractN/A

neuroscience↗

bioRxiv2019

Cation-chloride cotransporters and the polarity of GABA signaling in mouse hippocampal parvalbumin interneurons

Transmembrane chloride gradients govern the efficacy and polarity of GABA signaling in neurons and are usually maintained by the activity of cation chloride cotransporters, such as KCC2 and NKCC1. Whereas their role is well established in cortical principal neurons, it remains poorly documented in GABAergic interneurons. We used complementary electrophysiological approaches to compare the effects of GABAAR activation in adult mouse hippocampal parvalbumin interneurons (PV INs) and pyramidal cells (PCs). Loose cell attached, tight-seal and gramicidin-perforated patch recordings all show GABAAR-mediated transmission is slightly depolarizing and yet inhibitory in both PV INs and PCs. Focal GABA uncaging in whole-cell recordings reveal that KCC2 and NKCC1 are functional in both PV INs and PCs but differentially contribute to transmembrane chloride gradients in their soma and dendrites. Blocking KCC2 function depolarizes the reversal potential of GABAAR-mediated currents in PV INs and PCs, often beyond firing threshold, showing KCC2 is essential to maintain the inhibitory effect of GABAARs. Finally, we show that repetitive 10 Hz activation of GABAARs in both PV INs and PCs leads to a progressive decline of the postsynaptic response independently of the ion flux direction or KCC2 function. This suggests intraneuronal chloride buildup may not predominantly contribute to activity-dependent plasticity of GABAergic synapses in this frequency range. Altogether our data demonstrate similar mechanisms of chloride regulation in mouse hippocampal PV INs and PCs and suggest KCC2 downregulation in the pathology may affect the valence of GABA signaling in both cell types.\n\nKey point summaryO_LICation-chloride cotransporters (CCCs) play a critical role in controlling the efficacy and polarity of GABAA receptor (GABAAR)-mediated transmission in the brain, yet their expression and function in GABAergic interneurons has been overlooked.\nC_LIO_LIWe compared the polarity of GABA signaling and the function of CCCs in mouse hippocampal pyramidal neurons and parvalbumin-expressing interneurons.\nC_LIO_LIUnder resting conditions, GABAAR activation was mostly depolarizing and yet inhibitory in both cell types. KCC2 blockade further depolarized the reversal potential of GABAAR-mediated currents often above action potential threshold.\nC_LIO_LIHowever, during repetitive GABAAR activation, the postsynaptic response declined independently of the ion flux direction or KCC2 function, suggesting intracellular chloride buildup is not responsible for this form of plasticity.\nC_LIO_LIOur data demonstrate similar mechanisms of chloride regulation in mouse hippocampal pyramidal neurons and parvalbumin interneurons.\nC_LI

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