Glycinergic Transmission in the Presence and Absence of Functional GlyT2: Lessons From the Auditory Brainstem

Abstract

Synaptic transmission is controlled by re-uptake systems that reduce transmitter concentrations in the synaptic cleft and recycle the transmitter into presynaptic terminals. The re-uptake systems are thought to ensure cytosolic concentrations in the terminals that are sufficient for reloading empty synaptic vesicles (SVs). Genetic deletion of glycine transporter 2 (GlyT2) results in severely disrupted inhibitory neurotransmission and ultimately to death. Here we investigated the role of GlyT2 at inhibitory glycinergic synapses in the mammalian auditory brainstem. These synapses are tuned for resilience, reliability, and precision, even during sustained high-frequency stimulation when endocytosis and refilling of SVs probably contribute substantially to efficient replenishment of the readily releasable pool (RRP). Such robust synapses are formed between MNTB and LSO neurons (medial nucleus of the trapezoid body, lateral superior olive). By means of patch-clamp recordings, we assessed the synaptic performance in controls, in GlyT2 knockout mice (KOs), and upon acute pharmacological GlyT2 blockade. Via computational modeling, we calculated the reoccupation rate of empty release sites and RRP replenishment kinetics during 60-s challenge and 60-s recovery periods. Control MNTB-LSO inputs maintained high fidelity neurotransmission at 50 Hz for 60 s and recovered very efficiently from synaptic depression. During 'marathon-experiments' (30,600 stimuli in 20 min), RRP replenishment accumulated to 1,260-fold. In contrast, KO inputs featured severe impairments. For example, the input number was reduced to ~1 (vs. ~4 in controls), implying massive functional degeneration of the MNTB-LSO microcircuit and a role of GlyT2 during synapse maturation. Surprisingly, neurotransmission did not collapse completely in KOs as inputs still replenished their small RRP 80-fold upon 50 Hz | 60 s challenge. However, they totally failed to do so for extended periods. Upon acute pharmacological GlyT2 inactivation, synaptic performance remained robust, in stark contrast to KOs. RRP replenishment was 865-fold in marathon-experiments, only ~1/3 lower than in controls. Collectively, our empirical and modeling results demonstrate that GlyT2 re-uptake activity is not the dominant factor in the SV recycling pathway that imparts indefatigability to MNTB-LSO synapses. We postulate that additional glycine sources, possibly the antiporter Asc-1, contribute to RRP replenishment at these high-fidelity brainstem synapses.

Synergistic Control of Transmitter Turnover at Glycinergic Synapses by GlyT1, GlyT2, and ASC-1

“…This, however, requires a long administration of the drug and, e.g., persisting stimulation of the release [ 60 ]. In other experimental designs, a similar reduction of IPSC amplitude was not observed [ 67 , 70 ]. In some studies, a tonic inward current was induced by GlyT2 blocker application [ 60 , 70 ], which was absent in recordings from slices of GlyT2 −/− mice [ 61 , 65 ].…”

“…A recent study suggested that in GlyT2-KO mice, a subset of synaptic vesicles at glycinergic synapses has normal glycine content, at least at the synapse between the medial nucleus of the trapezoid body and the lateral superior olive (MNTB–LSO synapse) [ 67 ]. In this case, similar to previous findings [ 61 ], both sIPSC amplitude and frequency were significantly reduced.…”

“…Despite this discrepancy, it is apparent that the loss of GlyT2 does not completely prevent some vesicular loading of glycine [ 61 , 65 , 67 ]. Possible sources for this residual glycine are locally synthesized glycine by SHMT, but also other transporters that accept glycine as a substrate, such as some transporters of the SNAT family [ 68 ] or the ASC-1 transporter [ 69 ].…”

“…Interestingly, acute pharmacological blockade of GlyT2 by ALX1393 (2 µM) exerted a small effect on the time course of synaptic depression during high-frequency stimulation in the lateral superior olive (LSO; Brill et al 2021). While this observation could be explained by the existence of a large reserve pool of glycine-pre-filled vesicles in LSO glycinergic neurons, the significant slowing of recovery from depression, however, underlines the acute effect of GyT2 blockade on providing substrates for VIAAT-dependent vesical refilling [ 67 ].…”

Stochastic analysis of frequency bandwidth and noise attenuation in neurotransmission

“…Assuming all docking sites are filled initially n 0 = M , then presynaptic stimulation will cause a decrease in n(t) over time, and this vesicle depletion has often been referred to, in the literature, as synaptic depression [43], [44]. Fitting (8) to experimental data measuring the number of released vesicles over time upon presynaptic stimulation with a fixed frequency has been used to infer parameters across diverse synapses [45], [46]. Similar to (7), the differential equation describing the dynamics of the second-order moment is obtained as…”