Rapid, Activity-Independent Turnover of Vesicular Transmitter Content at a Mixed Glycine/GABA Synapse

Abstract

The release of neurotransmitter via the fusion of transmitter-filled, presynaptic vesicles is the primary means by which neurons relay information. However, little is known regarding the molecular mechanisms that supply neurotransmitter destined for vesicle filling, the endogenous transmitter concentrations inside presynaptic nerve terminals or the dynamics of vesicle refilling after exocytosis. We addressed these issues by recording from synaptically-coupled pairs of glycine/GABA co-releasing interneurons (cartwheel cells) of the mouse dorsal cochlear nucleus. We find that the plasma membrane transporter GlyT2 and the intracellular enzyme glutamate decarboxylase supply the majority of glycine and GABA, respectively. Pharmacological block of GlyT2 or glutamate decarboxylase led to rapid and complete rundown of transmission, whereas increasing GABA synthesis via intracellular glutamate uncaging dramatically potentiated GABA release within one minute. These effects were surprisingly independent of exocytosis, indicating that pre-filled vesicles re-equilibrated upon acute changes in cytosolic transmitter. Titration of cytosolic transmitter with postsynaptic responses indicated that endogenous, non-vesicular glycine/GABA levels in nerve terminals are 5 to 7 mM, and that vesicular transport mechanisms are not saturated under basal conditions. Thus, cytosolic transmitter levels dynamically set the strength of inhibitory synapses in a release-independent manner.

Unique pH dynamics in GABAergic synaptic vesicles illuminates the mechanism and kinetics of GABA loading

“…In support of this view, a study of peripheral cholinergic synapses indicated that inhibition of the vesicular ACh transporter did not reduce vesicular content in the absence of stimulation (42). In contrast, at glycine/GABA coreleasing interneuron varicosities, the vesicular GABA content is depleted by blocking cytosolic GABA synthesis, independent of exocytosis (43). This leaky nature of GABA through the lipid bilayer was also shown in proteoliposomes (10).…”

Chemical synaptic transmission onto superficial stellate cells of the mouse dorsal cochlear nucleus

“…Interestingly, in vivo work has suggested that GABAergic inhibition also controls the response properties of cartwheel and fusiform cells (Davis and Young 2000), and a recent in vitro study suggested that GABAergic inhibition controls the spread of parallel fiber excitation in the DCN (Middleton et al 2011). Since cartwheel cell transmission is predominantly glycinergic (Apostolides and Trussell 2013a;Golding and Oertel 1997;Mancilla and Manis 2009;Roberts et al 2008), these results suggest that stellate cells are a likely source of GABAergic inhibition.…”

Molecular Basis of the Dominant Negative Effect of a Glycine Transporter 2 Mutation Associated with Hyperekplexia

“…The neuronal GlyT2 transporter is involved in the removal and recycling of glycine from inhibitory synapses, generating a flux from the synaptic cleft to the presynaptic terminal and supplying substrate to the low affinity vesicular inhibitory amino acid transporter (2,3). Therefore, the synaptic glycine taken up by GlyT2 is the main source of the releasable transmitter at glycinergic synapses (4,5). Accordingly, inactivation of the mouse GlyT2 gene generates a complex postnatal neuromotor phenotype that mimics clinical signs of human hyperekplexia (2).…”