Inhibitory Synapse Formation in a Co-culture Model Incorporating GABAergic Medium Spiny Neurons and HEK293 Cells Stably Expressing GABA_A Receptors

Abstract

Inhibitory neurons act in the central nervous system to regulate the dynamics and spatio-temporal co-ordination of neuronal networks. GABA (γ-aminobutyric acid) is the predominant inhibitory neurotransmitter in the brain. It is released from the presynaptic terminals of inhibitory neurons within highly specialized intercellular junctions known as synapses, where it binds to GABA A receptors (GABA A Rs) present at the plasma membrane of the synapse-receiving, postsynaptic neurons. Activation of these GABA-gated ion channels leads to influx of chloride resulting in postsynaptic potential changes that decrease the probability that these neurons will generate action potentials.During development, diverse types of inhibitory neurons with distinct morphological, electrophysiological and neurochemical characteristics have the ability to recognize their target neurons and form synapses which incorporate specific GABA A Rs subtypes. This principle of selective innervation of neuronal targets raises the question as to how the appropriate synaptic partners identify each other.To elucidate the underlying molecular mechanisms, a novel in vitro co-culture model system was established, in which medium spiny GABAergic neurons, a highly homogenous population of neurons isolated from the embryonic striatum, were cultured with stably transfected HEK293 cell lines that express different GABA A R subtypes. Synapses form rapidly, efficiently and selectively in this system, and are easily accessible for quantification. Our results indicate that various GABA A R subtypes differ in their ability to promote synapse formation, suggesting that this reduced in vitro model system can be used to reproduce, at least in part, the in vivo conditions required for the recognition of the appropriate synaptic partners and formation of specific synapses. Here the protocols for culturing the medium spiny neurons and generating HEK293 cells lines expressing GABA A Rs are first described, followed by detailed instructions on how to combine these two cell types in co-culture and analyze the formation of synaptic contacts.

γ-Aminobutyric Acid Type A (GABAA) Receptor Subunits Play a Direct Structural Role in Synaptic Contact Formation via Their N-terminal Extracellular Domains

“…Because of a large number of proteins postulated to mediate these processes and the sheer complexity of their possible molecular interactions, exactly how these complex inter-neuronal connections are formed is often studied using reduced in vitro co-culture systems. Although far from the situation in vivo and subject to all the caveats that should surround any study in a reduced system, this approach has allowed us to establish that GABA A Rs have the ability to initiate the adhesion of GABAergic nerve terminals and formation of structurally and functionally competent GABAergic inhibitory synapses ( 33 , 53 ). These in vitro findings are supported by the in vivo evidence from GABA A α1 and α2 subunit knock-out mice demonstrating that the lack of these subunits in the hippocampus leads to prominent structural changes in specific types of inhibitory synapses ( 54 ).…”

Physiological and pharmacological properties of inhibitory postsynaptic currents mediated by α5β1γ2, α5β2γ2 and α5β3γ2 GABA A receptors

“…The study of individual GABA A R isoforms in neurons is confounded by the large number of GABA A R isoforms present and the unknown or poor selectivity profiles of available pharmacological blockers. This problem can be circumvented via the use of a neuron-HEK293 cell co-culture system, whereby functional 'heterosynapses' can be induced to form between neuronal presynaptic terminals and HEK293 cells that recombinantly express the GABA A R isoform of interest (Brown et al, 2014;Dixon et al, 2015;Dong et al, 2007). Using such a system, we investigated the biophysical properties of IPSCs mediated by recombinant α5β1γ2L, α5β2γ2L and α5β3γ2L GABA A Rs and characterized their sensitivity to four widely-used α5-specific inverse agonists: TB-21007, MRK-016, α5IA and L-655708.…”

SAHA (Vorinostat) Corrects Inhibitory Synaptic Deficits Caused by Missense Epilepsy Mutations to the GABAA Receptor γ2 Subunit

“…Although recombinantly expressing GABA A Rs in a heterologous expression system (e.g., HEK293 cells) allows individual isoforms to be studied in isolation, GABA must be artificially applied and thus it cannot reliably mimic the dynamic GABA concentration profile that exists in a synapse. Both problems can be solved simultaneously via the generation of “artificial synapses” between neurons and HEK293 cells that express the GABA A R isoform of interest (Dong et al, 2007 ; Brown et al, 2014 ; Dixon et al, 2015b ). By generating GABAergic synapses that incorporate defined subunit combinations, it is possible to determine how a given epilepsy-causing GABA A R mutation disrupts synaptic function.…”