Neuroligin-2 accelerates GABAergic synapse maturation in cerebellar granule cells.
Neuroligins (NLGs) are postsynaptic cell adhesion molecules that are thought to function in synaptogenesis. To investigate the role of NLGs on synaptic transmission once the synapse is formed, we transfected neuroligin-2 (NLG-2) in cultured mouse cerebellar granule cells (CGCs), and recorded GABA(A) (gamma-aminobutyric acid) receptor mediated miniature postsynaptic currents (mIPSCs). NLG-2 transfected cells had mIPSCs with faster decay than matching GFP expressing controls at young culture ages (days in vitro, DIV7-8). Down-regulation of NLG-2 by the isoform specific shRNA-NLG-2 resulted in an opposite effect. We and others have shown that the switch of alpha subunits of GABA(A)Rs from alpha2/3 to alpha1 underlies developmental speeding of the IPSC decay in various CNS regions, including the cerebellum. To assess whether the reduced decay time of mIPSCs by NLG-2 is due to the recruitment of more alpha1 containing GABA(A)Rs at the synapses, we examined the prolongation of current decay by the Zolpidem, which has been shown to preferentially enhance the activity of alpha1 subunit-containing GABA channel. The application of Zolpidem resulted in a significantly greater prolongation kinetics of synaptic currents in NLG-2 over-expressing cells than control cells, suggesting that NLG-2 over-expression accelerates synapse maturation by promoting incorporation of the alpha1 subunit-containing GABA(A)Rs at postsynaptic sites in immature cells. In addition, the effect of NLG-2 on the speeding of decay time course of synaptic currents was abolished when we used CGC cultures from alpha1-/- mice. Lastly, to exclude the possibility that the fast decay of mIPSCs induced by NLG-2 could be also due to the impacts of NLG-2 on the GABA transient in synaptic cleft, we measured the sensitivity of mIPSCs to the fast-off competitive antagonists TPMPA. We found that TPMPA similarly inhibits mIPSCs in control and NLG-2 over-expressing CGCs both at young age (DIV8) and old age (DIV14) of cultures. However, we confirm our previous finding of a greater inhibition of mIPSCs in young (DIV8) than more mature (DIV14) cultures. Together, our results suggest that NLG-2 does not alter uniquantal GABA release, and the fast decay of mIPSC induced by NLG-2 is due to the differential expression of postsynaptic GABA(A) receptor subtypes. Taken all together, we propose that NLG-2 plays important functional role in inhibitory synapse development and maturation.
Fu
Zhanyan
Z
Department of Psychiatry, Box 3209, Duke University Medical Center, 401I Bryan Research Building, Research Drive, Durham, NC 27710, USA. zhanyan.fu@duke.edu
Vicini
Stefano
S
D000818:Animals; D015816:Cell Adhesion Molecules, Neuronal; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology*; D018920:Coculture Techniques / Q000379:methods; D018755:GABA Agonists / Q000494:pharmacology; D018756:GABA Antagonists / Q000494:pharmacology; D005968:Glutamate Decarboxylase / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D053444:Inhibitory Postsynaptic Potentials / Q000187:drug effects / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics / Q000502:physiology*; D051379:Mice; D008822:Mice, Transgenic; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000502:physiology*; D009474:Neurons / Q000502:physiology*; D018408:Patch-Clamp Techniques / Q000379:methods; D010721:Phosphinic Acids / Q000494:pharmacology; D021122:Protein Subunits / Q000235:genetics; D011725:Pyridines / Q000494:pharmacology; D034622:RNA Interference / Q000502:physiology; D011963:Receptors, GABA-A / Q000172:deficiency / Q000235:genetics; D013569:Synapses / Q000502:physiology*; D014162:Transfection / Q000379:methods; D000077334:Zolpidem; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*
C101879:(1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid; D015816:Cell Adhesion Molecules, Neuronal; D018755:GABA Agonists; D018756:GABA Antagonists; C546240:GABRA1 protein, human; C546241:Gabra1 protein, mouse; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D010721:Phosphinic Acids; D021122:Protein Subunits; D011725:Pyridines; D011963:Receptors, GABA-A; C485184:enhanced green fluorescent protein; C098003:neuroligin 2; D049452:Green Fluorescent Proteins; D005680:gamma-Aminobutyric Acid; D000077334:Zolpidem; D005968:Glutamate Decarboxylase; C401141:glutamate decarboxylase 2