Functional reconstitution of glycinergic synapses incorporating defined glycine receptor subunit combinations

Zhang, Yan; Dixon, Curt B.; Keramidas, Angelo; Lynch, Joseph W.

doi:10.1016/j.neuropharm.2014.10.026

Cited by 29 publications

(61 citation statements)

References 58 publications

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“…b mutant GlyRs exhibited accelerated IPSC decay rates that were quantitatively similar to those recorded in native synapses in mutant mice homozygous for the same mutations (21). The main trends in our artificial synapse data are also supported by the fast agonist application experiments (Fig.…”

Section: A52ssupporting

confidence: 82%

“…This resulted in the expression of a1b heteromeric GlyRs (21). For artificial synapse experiments, mouse neuroligin-2A (pNice) and rat gephyrin (pCIS) were co-transfected along with GlyR plasmids to facilitate the formation of artificial synapses.…”

Section: Methodsmentioning

confidence: 99%

“…We have recently developed an 'artificial' synapse system that allows control over the subunit composition of GlyRs in glycinergic synapses (21). We inserted heteromeric a1b GlyRs incorporating each mutation in turn to evaluate the properties of the inhibitory postsynaptic currents (IPSCs) mediated by each mutant isoform.…”

Section: _____________________________________mentioning

confidence: 99%

“…Neurons were used in co-culture experiments between 3 -6 wk later (for GABAergic co-cultures) or 1 -4 wk later (for glycinergic co-cultures). respectively (Zhang et al, 2014). When expressing a1b2g2 GABA A Rs, the a1, b2, g2, EGFP and neuroligin 2A plasmid DNAs should be transfected in a 1:1:4:1:1 ratio with a combined total of 0.5 µg DNA.…”

Section: Preparation Of Neuron Culturesmentioning

confidence: 99%

“…They have also been employed to characterise the functional properties of inhibitory post-synaptic currents (IPSCs), and have revealed kinetic differences among different GABA A R isoforms (Wu et al, 2012; and GlyR isoforms (Zhang et al, 2014).…”

Section: Introductionmentioning

confidence: 99%

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Physiological properties of Glycinergic synapses with defined subunit compositions

Zhang¹

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Glycine receptors (GlyRs) mediate fast inhibitory neurotransmission in the spinal cord and brainstem. Four GlyR subunits (α1 -3, β) have been identified in humans, and their differential anatomical distributions result in a diversity of synaptic isoforms with unique physiological and pharmacological properties. However, despite their importance in controlling neuronal function, little is known about these properties. To address this, we developed an 'artificial' synapse system which allows to control over the synaptic GlyR subunit composition. We induced the formation of recombinant synapses between cultured spinal neurons and HEK293 cells expressing GlyR subunits of interest plus the synapse-promoting molecule, neuroligin-2A. In the heterosynapses thus formed, recombinant α1β and α3β GlyRs mediated fast decaying inhibitory postsynaptic currents (IPSCs) whereas α2β GlyRs mediated slow decaying IPSCs. These results are consistent with the fragmentary information available from native synapses and single channel kinetic studies. As β subunit incorporation is considered essential for localizing GlyRs at the synapse, we were surprised that α1 -3 homomers supported robust IPSCs with β subunit incorporation accelerating IPSC rise and decay times in α2β and α3β heteromers only. Finally, heterosynapses incorporating α1 D80A β and α1 A52S β GlyRs exhibited accelerated IPSC decay rates closely resembling those recorded in native synapses from mutant mice homozygous for these mutations, providing an additional validation of our technique. As our model system successfully recapitulates the effects of known GlyR disease mutations, we employed it to investigate the effects of new GlyR disease mutations.Hyperekplexia is a human neuromotor disorder caused by mutations that impair glycinergic neurotransmission. We investigated the mechanism by which gain-of-function GlyR mutations cause hyperekplexia. We identified two new gain-of-function mutations (I43F, W170S) and characterised these along with the known gain-of-function mutations (Q226E, V280M, R414H) to identify how they cause hyperekplexia. Using 'artificial' synapses, we show that all mutations prolong the decay of IPSCs and induce spontaneous activation. As these effects may deplete the chloride electrochemical gradient, hyperekplexia could potentially result from reduced glycinergic inhibitory efficacy. However, we consider this unlikely as it should also lead to pain sensitization and a hyperekplexia phenotype that correlates with mutation severity, neither of which is observed in patients. We also rule out small increases in IPSC decay times (as caused by W170S and R414H)as a possible mechanism given that the clinically-important drug, tropisetron, increases glycinergic IPSC decay times without causing motor side effects. A recent study concluded that an elevated intracellular chloride concentration late during development ablates α1β glycinergic synapses but iii spares GABAergic synapses. As this mechanism satisfies all our considerations, we propose it is pr...

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Section: A52ssupporting

confidence: 82%

Section: Methodsmentioning

confidence: 99%

Section: _____________________________________mentioning

confidence: 99%

Section: Preparation Of Neuron Culturesmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Physiological properties of Glycinergic synapses with defined subunit compositions

Zhang¹

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Glycine Receptor Drug Discovery

Lynch

Zhang

Talwar

et al. 2017

Advances in Pharmacology

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Gephyrin and the regulation of synaptic strength and dynamics at glycinergic inhibitory synapses

Álvarez

2017

Brain Research Bulletin

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Glycinergic synapses predominate in brainstem and spinal cord where they modulate motor and sensory processing. Their postsynaptic mechanisms have been considered rather simple because they lack a large variety of glycine receptor isoforms and have relatively simple postsynaptic densities at the ultrastructural level. However, this simplicity is misleading being their postsynaptic regions regulated by a variety of complex mechanisms controlling the efficacy of synaptic inhibition. Early studies suggested that glycinergic inhibitory strength and dynamics depend largely on structural features rather than on molecular complexity. These include regulation of the number of postsynaptic glycine receptors, their localization and the amount of co-localized GABA receptors and GABA-glycine co-transmission. These properties we now know are under the control of gephyrin. Gephyrin is the first postsynaptic scaffolding protein ever discovered and it was recently found to display a large degree of variation and regulation by splice variants, posttranslational modifications, intracellular trafficking and interactions with the underlying cytoskeleton. Many of these mechanisms are governed by converging excitatory activity and regulate gephyrin oligomerization and receptor binding, the architecture of the postsynaptic density (and by extension the whole synaptic complex), receptor retention and stability. These newly uncovered molecular mechanisms define the size and number of gephyrin postsynaptic regions and the numbers and proportions of glycine and GABA receptors contained within. All together, they control the emergence of glycinergic synapses of different strength and temporal properties to best match the excitatory drive received by each individual neuron or local dendritic compartment.

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Functional reconstitution of glycinergic synapses incorporating defined glycine receptor subunit combinations

Cited by 29 publications

References 58 publications

Physiological properties of Glycinergic synapses with defined subunit compositions

Physiological properties of Glycinergic synapses with defined subunit compositions

Glycine Receptor Drug Discovery

Gephyrin and the regulation of synaptic strength and dynamics at glycinergic inhibitory synapses

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