Refine
Document Type
- Article (1)
- Doctoral Thesis (1)
Language
- English (2) (remove)
Has Fulltext
- yes (2) (remove)
Keywords
- GAT-1 (1)
- GAT-3 (1)
- GlyT1 (1)
- Hippocampus (1)
- Inferior colliculus (1)
Faculty / Organisational entity
Glycine constitutes the major neurotransmitter at inhibitory synapses of lower brain regions.
A rapid removal of glycine from the synaptic cleft and consequent recycling is crucial for
synaptic transmission in systems with high effort on temporal precision. This is mainly
achieved by glycine translocation via two glycine transporters (GlyTs), namely GlyT1 and
GlyT2. At inhibitory synapses, GlyT2 was found to be specifically expressed by neurons,
supplying the presynapse with glycine needed for vesicle filling. In contrast, GlyT1 is attributed
to astrocytes and primarily mediates the termination of synaptic transmission by glycine
removal from the synaptic cleft. Employing patch-clamp recordings from principal neurons of
the lateral superior olive (LSO) in acute brainstem slices of GlyT1b/c knockout (KO) mice and
wildtype (WT) littermates at postnatal day 20, I analyzed how postsynaptic responses are
changed in a GlyT1-depleted environment. During spontaneous vesicle release I found no
change of postsynaptic responses, contradicting my initial hypothesis of prolonged decay
times. Electrical stimulation of fibers of the medial nucleus of the trapezoid body (MNTB),
which are known to form fast, reliable and highly precise synapses with LSO principal neurons,
revealed that GlyT1 is involved in proper synaptic function during sustained, high frequent
synaptic transmission. Stimulation with 50 Hz led to a stronger decay time and latency
prolongation in GlyT1b/c KO, accelerating to 60% longer decay times and 30% longer latencies.
Additionally, a more pronounced frequency-dependent depression and fidelity decrease was
observed during stimulation with 200 Hz in GlyT1b/c KO, resulting in 67% smaller amplitudes
and only 25% of WT fidelity at the end of the challenge. Basic properties like readily releasable
pool, release probability, and quantal size (q) were not altered in GlyT1b/c KO, but
interestingly q decreased during 50 Hz and 100 Hz challenges to about 84%, which was not
observed in WT. I conclude that stronger accumulation of extracellular glycine due to GlyT1
loss leads to prolonged activation of postsynaptic glycine receptors (GlyRs). As a further
consequence, activation of presynaptic GlyRs in the vicinity of the synaptic cleft might be
enhanced, accompanied by a stronger occurrence of shunting inhibition. Furthermore, I
assume a GlyT1-dependent glycine shuttle, which is absent at GlyT1b/c KO synapses. This
could result in a diminished glycine supply to GlyT2 located at more distant sites, causing a
disturbed replenishment during periods with excess release of glycine. Conclusively, my study
reveals a contribution of astrocytes in fast and reliable synaptic transmission at the MNTB-LSO
synapse, which in turn is crucial for proper sound source localization.
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.