Development of gamma-aminobutyric acidergic synapses in cultured hippocampal neurons.
The formation and maturation of gamma-aminobutyric acid (GABA)-ergic synapses was studied in cultured hippocampal pyramidal neurons by both performing immunocytochemistry for GABAergic markers and recording miniature inhibitory postsynaptic currents (mIPSCs). Nascent GABAergic synapses appeared between 3 and 8 days in vitro (DIV), with GABAA receptor subunit clusters appearing first, followed by GAD-65 puncta, then functional synapses. The number of GABAergic synapses increased from 7 to 14 DIV, with a corresponding increase in frequency of mIPSCs. Moreover, these new GABAergic synapses formed on neuronal processes farther from the soma, contributing to decreased mIPSC amplitude and slowed mIPSC 19-90% rise time. The mIPSC decay quickened from 7 to 14 DIV, with a parallel change in the distribution of the alpha5 subunit from diffuse expression at 7 DIV to clustered expression at 14 DIV. These alpha5 clusters were mostly extrasynaptic. The alpha1 subunit was expressed as clusters in none of the neurons at 7 DIV, in 20% at 14 DIV, and in 80% at 21 DIV. Most of these alpha1 clusters were expressed at GABAergic synapses. In addition, puncta of GABA transporter 1 (GAT-1) were localized to GABAergic synapses at 14 DIV but were not expressed at 7 DIV. These studies demonstrate that mIPSCs appear after pre- and postsynaptic elements are in place. Furthermore, the process of maturation of GABAergic synapses involves increased synapse formation at distal processes, expression of new GABAA receptor subunits, and GAT-1 expression at synapses; these changes are reflected in altered frequency, kinetics, and drug sensitivity of mIPSCs.
Neuroscience Graduate Program, University of Virginia, Charlottesville, Virginia 22908, USA.
Catherine Croft
CC
Swanwick
Namita R
NR
Murthy
Zakaria
Z
Mtchedlishvili
Werner
W
Sieghart
Jaideep
J
Kapur
D000818:Animals; D000831:Animals, Newborn; D015415:Biomarkers / Q000378:metabolism; D002478:Cells, Cultured; D004594:Electrophysiology; D005260:Female; D050485:GABA Plasma Membrane Transport Proteins / Q000378:metabolism; D006624:Hippocampus / Q000166:cytology* / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D011247:Pregnancy; D021122:Protein Subunits / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011963:Receptors, GABA-A / Q000378:metabolism; D013569:Synapses / Q000378:metabolism*; D009435:Synaptic Transmission / Q000502:physiology; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*
D015415:Biomarkers; D050485:GABA Plasma Membrane Transport Proteins; D021122:Protein Subunits; D011963:Receptors, GABA-A; D005680:gamma-Aminobutyric Acid