Development of gamma-aminobutyric acidergic synapses in cultured hippocampal neurons

J Comp Neurol. 2006 Apr 10;495(5):497-510. doi: 10.1002/cne.20897.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Animals, Newborn
  • Biomarkers / metabolism
  • Cells, Cultured
  • Electrophysiology
  • Female
  • GABA Plasma Membrane Transport Proteins / metabolism
  • Hippocampus / cytology*
  • Hippocampus / metabolism
  • Neurons / cytology
  • Neurons / metabolism*
  • Pregnancy
  • Protein Subunits / metabolism
  • Rats
  • Rats, Sprague-Dawley
  • Receptors, GABA-A / metabolism
  • Synapses / metabolism*
  • Synaptic Transmission / physiology
  • gamma-Aminobutyric Acid / metabolism*


  • Biomarkers
  • GABA Plasma Membrane Transport Proteins
  • Protein Subunits
  • Receptors, GABA-A
  • gamma-Aminobutyric Acid