Abstract
The corelease of several neurotransmitters from a single synaptic vesicle has been observed at many central synapses. Nevertheless, the signaling synergy offered by cotransmission and the mechanisms that maintain the optimal release and detection of neurotransmitters at mixed synapses remain poorly understood, thus limiting our ability to interpret changes in synaptic signaling and identify molecules important for plasticity. In the brainstem and spinal cord, GABA and glycine cotransmission is facilitated by a shared vesicular transporter VIAAT (also named VGAT), and occurs at many immature inhibitory synapses. As sensory and motor networks mature, GABA/glycine cotransmission is generally replaced by either pure glycinergic or GABAergic transmission, and the functional role for the continued corelease of GABA and glycine is unclear. Whether or not, and how, the GABA/glycine content is balanced in VIAAT-expressing vesicles from the same terminal, and how loading variability effects the strength of inhibitory transmission is not known. Here, we use a combination of loose-patch (LP) and whole-cell (WC) electrophysiology in cultured spinal neurons of GlyT2:eGFP mice to sample miniature inhibitory post synaptic currents (mIPSCs) that originate from individual GABA/glycine co-releasing synapses and develop a modeling approach to illustrate the gradual change in mIPSC phenotypes as glycine replaces GABA in vesicles. As a consistent GABA/glycine balance is predicted if VIAAT has access to both amino-acids, we test whether vesicle exocytosis from a single terminal evokes a homogeneous population of mixed mIPSCs. We recorded mIPSCs from 18 individual synapses and detected glycine-only mIPSCs in 4/18 synapses sampled. The rest (14/18) were co-releasing synapses that had a significant proportion of mixed GABA/glycine mIPSCs with a characteristic biphasic decay. The majority (9/14) of co-releasing synapses did not have a homogenous phenotype, but instead signaled with a combination of mixed and pure mIPSCs, suggesting that there is variability in the loading and/or storage of GABA and glycine at the level of individual vesicles. Our modeling predicts that when glycine replaces GABA in synaptic vesicles, the redistribution between the peak amplitude and charge transfer of mIPSCs acts to maintain the strength of inhibition while increasing the temporal precision of signaling.
Highlights
A well-established and long accepted postulate known as ‘‘Dale’s principle’’ proposes that neurons have chemical unity for signaling (Strata and Harvey, 1999; Tritsch et al, 2016) —meaning that they communicate by releasing the same neurotransmitter at all their synapses
LP-miniature inhibitory postsynaptic currents (mIPSCs) were never detected if the LP-pipette was not centered precisely on the GlyT2-eGFP+ varicosity, consistent with the prediction that the LP-pipette will detect less than 1% of currents that originate outside the LP pipette (Forti et al, 1997) and suggestive that detection of current from two independent synases is unlikely
We can excluded a significant contribution of the sniffer cell in the BON/HEK model because scanning across the entire sniffer HEK cell surface with brief iontophoretic applications of a fixed [GABA] and [glycine] evoked stable biphasic currents (Supplementary Figures S1D,E), again supporting heterogeneous vesicular content that favors glycineor GABA-like mIPSCs
Summary
A well-established and long accepted postulate known as ‘‘Dale’s principle’’ proposes that neurons have chemical unity for signaling (Strata and Harvey, 1999; Tritsch et al, 2016) —meaning that they communicate by releasing the same neurotransmitter at all their synapses. Biochemical examinations of GABA and glycine uptake into synaptosomes (an isolated synaptic terminal preparation) indicated that both amino acids compete for the same vesicular transporter (Fykse and Fonnum, 1988; Burger et al, 1991; Christensen and Fonnum, 1991). This idea was endorsed when VIAAT was found in both GABAergic and glycinergic terminals (McIntire et al, 1997; Sagné et al, 1997) and taken together, these data suggested that the vesicular content is determined by the relative occupancy of GABA/glycine at VIAAT (Gasnier, 2000) and will be primarily determined by the relative presynaptic cytosolic concentrations of the two inhibitory neurotransmitters
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