In vivo transgenic expression of collybistin in neurons of the rat cerebral cortex

Abstract

Collybistin (CB) is a guanine nucleotide exchange factor selectively localized to γ-aminobutyric acid (GABA)ergic and glycinergic postsynapses. Active CB interacts with gephyrin, inducing the submembranous clustering and the postsynaptic accumulation of gephyrin, which is a scaffold protein that recruits GABA receptors (GABA Rs) at the postsynapse. CB is expressed with or without a src homology 3 (SH3) domain. We have previously reported the effects on GABAergic synapses of the acute overexpression of CB or CB in cultured hippocampal (HP) neurons. In the present communication, we are studying the effects on GABAergic synapses after chronic in vivo transgenic expression of CB2 or CB2 in neurons of the adult rat cerebral cortex. The embryonic precursors of these cortical neurons were in utero electroporated with CB or CB DNAs, migrated to the appropriate cortical layer, and became integrated in cortical circuits. The results show that: 1) the strength of inhibitory synapses in vivo can be enhanced by increasing the expression of CB in neurons; and 2) there are significant differences in the results between in vivo and in culture studies. J. Comp. Neurol. 525:1291-1311, 2017. © 2016 Wiley Periodicals, Inc.

Mutation p.R356Q in the Collybistin Phosphoinositide Binding Site Is Associated With Mild Intellectual Disability

“…Previous studies in this neuronal expression system have shown that wild-type recombinant collybistin isoforms (e.g., CB2 SH3+ , CB2 SH3- , CB3 SH3+ , and CB3 SH3- ) target to and concentrate at GABAergic postsynapses (Chiou et al, 2011). Notably, isoforms lacking the SH3 domain (e.g., CB2 SH3- and CB3 SH3- ) induce the formation of synaptic gephyrin superclusters (Chiou et al, 2011; Fekete et al, 2017) that are accompanied by a significant increase in the amplitude of miniature inhibitory postsynaptic currents (mIPSCs). In this study, we utilized triple-label immunofluorescence with mouse anti-myc, rabbit anti-gephyrin and sheep anti-GAD to reveal exogenous wild-type and mutant collybistin, native gephyrin and GAD, respectively (Figures 3A–C).…”

“…Hence, we used CB3 SH3- rather than CB3 SH3+ in our PI3P pull-down and cellular clustering assays. In cultured hippocampal neurones, all tested collybistin isoforms (CB2 SH3+ , CB2 SH3- , CB3 SH3+ and CB3 SH3- ) target to and concentrate at GABAergic postsynapses (Chiou et al, 2011; Fekete et al, 2017) with the major difference being that transfection of de-regulated isoforms that lack the SH3 domain (e.g., CB3 SH3- ) results in large postsynaptic gephyrin and GABA A receptor superclusters, while isoforms containing the SH3 domain induce the formation of supernumerary non-synaptic clusters (Chiou et al, 2011; Fekete et al, 2017). In this system, overexpression of the myc-CB3 SH3- R356Q mutant resulted in a decrease in the overall number of synaptic gephyrin clusters compared to controls.…”

Benign Effect of Extremely Low‐Frequency Electromagnetic Field on Brain Plasticity Assessed by Nitric Oxide Metabolism during Poststroke Rehabilitation

“…On the other hand, NO causes a neurotransmission inhibition through gamma-aminobutyric acid- (GABA-) ergic synaptic communication. It is associated with ion exchange and regulation of membrane excitation [ 21 , 22 ]. Moreover, NO as an important vasodilation factor mediates neurovascular coupling.…”

Expression of protocadherin‐γC4 protein in the rat brain

“…The procedure has been described elsewhere (Fekete et al, ; Fekete et al, ; Li, Serwanski, Miralles, Nagata, & De Blas, ; Li et al, ). Briefly, free‐floating brain sections, prepared as described above from P35 SD rats, were incubated with 5% normal goat serum (NGS)/0.3% Triton X‐100/ 0.12 M PB for 1 hour at RT, followed by incubation for 2 days at 4°C in a mixture of primary antibodies raised in different species in 2%NGS/0.3% Triton X‐100 in 0.12 M PB at 4°C.…”