Abstract
In the adult brain GABAA receptors (GABAARs) mediate the majority of synaptic inhibition that provides inhibitory balance to excitatory drive and controls neuronal output. In the immature brain GABAAR signaling is critical for neuronal development. However, the cell-autonomous role of GABAARs in synapse development remains largely unknown. We have employed the CRISPR-CAS9 technology to genetically eliminate GABAARs in individual hippocampal neurons and examined GABAergic and glutamatergic synapses. We found that development of GABAergic synapses, but not glutamatergic synapses, critically depends on GABAARs. By combining different genetic approaches, we have also removed GABAARs and two ionotropic glutamate receptors, AMPA receptors (AMPARs) and NMDA receptors (NMDARs), in single neurons and discovered a striking dichotomy. Indeed, while development of glutamatergic synapses and spines does not require signaling mediated by these receptors, inhibitory synapse formation is crucially dependent on them. Our data reveal a critical cell-autonomous role of GABAARs in inhibitory synaptogenesis and demonstrate distinct molecular mechanisms for development of inhibitory and excitatory synapses.
Highlights
GABAA receptors (GABAARs) are ligand-gated hetero-pentameric anion channels assembled from various combinations of 19 subunits: α (1-6), β (1-3), γ (1-3), δ, θ, π, and ρ (1-3), most GABAARs in the brain consist of two α subunits, two β subunits, and one γ or δ subunit (Macdonald and Olsen, 1994; Chang et al, 1996; Sieghart and Sperk, 2002; Mody and Pearce, 2004; Olsen and Sieghart, 2008)
We have employed the CRISPR-Cas9 technology to develop single-guide RNAs to target gene loci of three GABRBs, respectively, that encode GABAAR β subunits in mouse genome
We found that compared with control neurons whereby GABAAR-mediated tonic currents were readily detected by bicuculline, little tonic currents were observed in neurons expressing β1-3 guidance RNA (gRNA) (Figure 1H)
Summary
GABAA receptors (GABAARs) are ligand-gated hetero-pentameric anion channels assembled from various combinations of 19 subunits: α (1-6), β (1-3), γ (1-3), δ, , θ, π, and ρ (1-3), most GABAARs in the brain consist of two α subunits, two β subunits, and one γ or δ subunit (Macdonald and Olsen, 1994; Chang et al, 1996; Sieghart and Sperk, 2002; Mody and Pearce, 2004; Olsen and Sieghart, 2008). Synaptic GABAARs in knockdown or conventional knockout (KO) mice of GABAAR subunits are reduced or lost, leading to the impairment of GABAergic synapse formation and maturation (Fritschy et al, 2006, 2012; Patrizi et al, 2008; Frola et al, 2013), which provides genetic evidence for the role of GABAAR subunits in synapse development In these genetic models, neurons may adapt to the global absence of the GABAAR subunits and to altered neural network activities throughout their development. The cell-autonomous role of GABAARs in the regulation of synapse development remains largely unclear
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