Type A GABA-Receptor-Dependent Synaptic Transmission Sculpts Dendritic Arbor Structure inXenopusTadpolesIn Vivo

Abstract

The emergence of dendritic arbor structure in vivo depends on synaptic inputs. We tested whether inhibitory GABAergic synaptic transmission regulates Xenopus optic tectal cell dendritic arbor development in vivo by expressing a peptide corresponding to an intracellular loop (ICL) of the ␥2 subunit of type A GABA receptors (GABA A R), which is required to anchor GABA A receptors to the postsynaptic scaffold. Enhanced green fluorescent protein (EGFP)-tagged ICL (EGFP-ICL) was distributed in a punctate pattern at putative inhibitory synapses, identified by vesicular GABA transporter immunoreactive puncta. ICL expression completely blocked GABA A R-mediated transmission in 36% of transfected neurons and significantly reduced GABA A R-mediated synaptic currents relative to AMPA receptor-mediated synaptic currents in the remaining transfected neurons without altering release probability or neuronal excitability. Further analysis of ICL-expressing neurons with residual GABA A R-mediated inputs showed that the capacity of benzodiazepine to enhance GABAergic synaptic responses was reduced in ICL-expressing neurons, indicating that they were likely depleted of ␥2 subunit-containing GABA A R. Neurons expressing a mutant form of ICL were comparable to controls. In vivo time-lapse images showed that ICL-expressing neurons have more sparsely branched dendritic arbors, which expand over larger neuropil areas than EGFPexpressing control neurons. Analysis of branch dynamics indicated that ICL expression affected arbor growth by reducing rates of branch addition. Furthermore, we found that decreasing GABAergic synaptic transmission with ICL expression blocked visual experience dependent dendritic arbor structural plasticity. Our findings establish an essential role for inhibitory GABAergic synaptic transmission in the regulation of dendritic structural plasticity in Xenopus in vivo.

GABAergic control of retinal ganglion cell dendritic development

“…Indeed, turning GABAergic excitation into inhibition by manipulating Cltransport activity during the period of dendritic growth strongly suppresses further growth. In agreement with our findings, a study on frog optic tectum has shown that when GABAergic synaptic activity is reduced by modifying the c2 subunit of GABA A receptors, tectal neurons have more sparsely branched dendritic arbors that expand over larger areas (Shen et al, 2009). It has been reported in cultures that spontaneous cholinergic nicotinic activity triggers the GABAergic shift from excitation to inhibition through KCC2 upregulation, resulting in dendritic pruning (Liu et al, 2006).…”

Inhibition to excitation ratio regulates visual system responses and behavior in vivo

“…A dual CMV promoter plasmid expressing enhanced green fluorescent protein (EGFP) and ICL, EGFP and mutant ICL (mICL), or EGFP alone was used for transfections. ICL was modified from the murine sequence of ICL (kindly provided by Dr. Simon Rumpel, The Institute for Molecular Pathology, Vienna, Austria) (Shen et al 2009). The mICL is generated by exchanging Ser (S) to Ala (A) of ICL (HYFVSNRKPSKDKDKKKKNPLLRMFSFKAPTIDIRPRSAT-IQMNNATHLQERDEEYGYECLDGKDCASFFCCFEDCRTGAW-RHGRIHIRIAKMDS) (Shen et al 2009).…”

“…ICL was modified from the murine sequence of ICL (kindly provided by Dr. Simon Rumpel, The Institute for Molecular Pathology, Vienna, Austria) (Shen et al 2009). The mICL is generated by exchanging Ser (S) to Ala (A) of ICL (HYFVSNRKPSKDKDKKKKNPLLRMFSFKAPTIDIRPRSAT-IQMNNATHLQERDEEYGYECLDGKDCASFFCCFEDCRTGAW-RHGRIHIRIAKMDS) (Shen et al 2009). The X. laevis cDNA of the GABA A R ␥2 subunit was cloned from a cDNA library made with the SMART rapid amplification of cDNA ends amplification kit (Clontech Laboratories, Mountain View, CA), according to the manufacturer's protocol, and sequenced.…”

“…For all recordings, the brains were perfused with extracellular saline containing (in mM): 115 NaCl, 2 KCl, 3 CaCl 2 , 1.5 MgCl 2 , 5 HEPES, 10 glucose, and 0.01 glycine, pH 7.2, osmolality 255 mOsm, throughout the recording. For recordings from neurons in isolated brains, tadpoles were anesthetized with 0.02% MS-222 (Sigma-Aldrich), brains were dissected, and neurons were recorded in whole-cell mode as described (Shen et al 2009). When required, TTX (1 M), DZ (10 M), 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione (NBQX; 20 M), and DL-2-amino-5-phosphonopentanoic acid (D,L-APV) (50 M, all Sigma-Aldrich) were included in the extracellular saline.…”

The Development of Developmental Neuroscience

“…Culture studies in the 80s showed how the cell forms an axon and by default, other processes become dendrites (Dotti et al, 1988). The influence of neurotrophins as well as synaptic inputs in shaping dendrite development continues to be shown beautifully in single-cell labeling and cell-perturbation studies (McAllister et al, 1999;Shen et al, 2009). Quite astonishing is that retinal ganglion cells display a huge variety of dendritic shapes (Kong et al, 2005).…”