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Abstract
Fast inhibitory neurotransmission in the brain is mediated by wide range of GABAA receptor (GABAAR) and glycine receptor (GlyR) isoforms, each with different physiological and pharmacological properties. Because multiple isoforms are expressed simultaneously in most neurons, it is difficult to define the properties of individual isoforms under synaptic stimulation conditions in vivo. Although recombinant expression systems permit the expression of individual isoforms in isolation, they require exogenous agonist application which cannot mimic the dynamic neurotransmitter profile characteristic of native synapses. We describe a neuron-HEK293 cell co-culture technique for generating inhibitory synapses incorporating defined combinations of GABAAR or GlyR subunits. Primary neuronal cultures, prepared from embryonic rat cerebral cortex or spinal cord, are used to provide presynaptic GABAergic and glycinergic terminals, respectively. When the cultures are mature, HEK293 cells expressing the subunits of interest plus neuroligin 2A are plated onto the neurons, which rapidly form synapses onto HEK293 cells. Patch clamp electrophysiology is then used to analyze the physiological and pharmacological properties of the inhibitory postsynaptic currents mediated by the recombinant receptors. The method is suitable for investigating the kinetic properties or the effects of drugs on inhibitory postsynaptic currents mediated by defined GABAAR or GlyR isoforms of interest, the effects of hereditary disease mutations on the formation and function of both types of synapses, and synaptogenesis and synaptic clustering mechanisms. The entire cell preparation procedure takes 2–5 weeks.
Highlights
The central nervous system is comprised of circuits of interconnected neurons that serve to process specific types of information
While we found that some co-cultures exhibited spontaneous activity in almost all green fluorescent HEK293 cells, it was more typical to observe spontaneous glycinergic inhibitory post-synaptic currents (IPSCs) in around 20% of cells
It is important to establish that the spontaneous IPSCs produced by the co-culture synapses exhibit similar characteristics to those mediated by native synapses incorporating the same subunits
Summary
The central nervous system is comprised of circuits of interconnected neurons that serve to process specific types of information. These circuits regulate their own output by feedback and feedforward connections. There are 19 GABAAR genes (α1–6, β1–3, γ1–3, δ, ε, θ, π, and ρ1–3) with the most common synaptic isoform comprising α1, β2, and γ2 subunits in a 2:2:1 stoichiometry. GlyRs exhibit far less diversity with only four genes (α1–3, β) in humans (Lynch, 2009) They belong to the pLGIC family and synaptic GlyR isoforms comprise heteromeric assemblies of α and β subunits in a 2:3 or 3:2 stoichiometry (Durisic et al, 2012; Yang et al, 2012)
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