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Neuronal inhibition is mediated by glycine and/or GABA. Inferior colliculus (IC) neurons receive glycinergic and GABAergic
inputs, whereas inhibition in hippocampus (HC) predominantly relies on GABA. Astrocytes heterogeneously
express neurotransmitter transporters and are expected to adapt to the local requirements regarding neurotransmitter
homeostasis. Here we analyzed the expression of inhibitory neurotransmitter transporters in IC and HC astrocytes using
whole-cell patch-clamp and single-cell reverse transcription-PCR. We show that most astrocytes in both regions expressed
functional glycine transporters (GlyTs). Activation of these transporters resulted in an inward current (IGly) that
was sensitive to the competitive GlyT1 agonist sarcosine. Astrocytes exhibited transcripts for GlyT1 but not for
GlyT2. Glycine did not alter the membrane resistance (RM) arguing for the absence of functional glycine receptors (GlyRs).
Thus, IGly was mainly mediated by GlyT1. Similarly, we found expression of functional GABA transporters (GATs) in all IC
astrocytes and about half of the HC astrocytes. These transporters mediated an inward current (IGABA) that was sensitive to
the competitive GAT-1 and GAT-3 antagonists NO711 and SNAP5114, respectively. Accordingly, transcripts for GAT-1 and
GAT-3 were found but not for GAT-2 and BGT-1. Only in hippocampal astrocytes, GABA transiently reduced
RM demonstrating the presence of GABAA receptors (GABAARs). However, IGABA was mainly not contaminated
by GABAAR-mediated currents as RM changes vanished shortly after GABA application. In both regions, IGABA
was stronger than IGly. Furthermore, in HC the IGABA/IGly ratio was larger compared to IC. Taken together, our
results demonstrate that astrocytes are heterogeneous across and within distinct brain areas. Furthermore, we
could show that the capacity for glycine and GABA uptake varies between both brain regions.
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.