Developmental changes of GABAergic synapses formed between primary cultured cortical neurons.
The characteristics of functional changes of GABAergic synapses between cultured rat cortical neurons were observed by monitoring intracellular calcium level ([Ca2+]in) during development in vitro. After 5 days in vitro (DIV), cultured cortical neurons spontaneously exhibited synchronous oscillatory changes in [Ca2+]in, which were derived from synaptic activity. Exposure to bicuculline, antagonist of gamma-aminobutyric acid (GABA)(A) receptors, caused a marked decrease in the frequency of [Ca2+]in oscillations at 7-20 DIV. Although the frequency of spontaneous oscillations increased during this culture period, the ratio of the decrease in the frequency following bicuculline treatment did not significantly change. Thereafter, to investigate the detailed morphological changes of GABAergic synapses during development in vitro, the cultured neurons were immunostained with antibodies to glutamic acid decarboxylase (GAD), synaptophysin and GABA(A) receptor and were observed under a confocal laser microscope. Most of the GAD-positive puncta colocalized with synaptophysin-positive puncta and were opposed to GABA(A) receptor-positive structures. The images of GAD-positive puncta were reconstructed from the confocal three-dimensional data to analyze their number, volume, and surface area. The number of these puncta increased with culture time at 7-20 DIV. Although the volume of individual GAD-positive puncta did not significantly change, the surface area decreased in a time-dependent manner over the culture period. This system that we developed enabled us to investigate in detail the morphological and functional changes of GABAergic synapses during neuronal development.
Kato-Negishi
Midori
M
Laboratory of Anatomy and Cell Biology, Department of Basic Technique and Facilities, Tokyo Metropolitan Institute for Neuroscience, Tokyo Metropolitan Organization for Medical Research, 2-6 Musashi-dai, Fuchu, Tokyo 183-8526, Japan. midori@tmin.ac.jp
Muramoto
Kazuyo
K
Kawahara
Masahiro
M
Kuroda
Yoichiro
Y
Ichikawa
Masumi
M
D000818:Animals; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000196:embryology* / Q000254:growth & development* / Q000378:metabolism; D018756:GABA Antagonists / Q000494:pharmacology; D058787:GABA-A Receptor Antagonists; D005968:Glutamate Decarboxylase / Q000378:metabolism; D007150:Immunohistochemistry; D018613:Microscopy, Confocal; D009434:Neural Pathways / Q000196:embryology* / Q000254:growth & development / Q000378:metabolism; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D011963:Receptors, GABA-A / Q000378:metabolism; D013569:Synapses / Q000187:drug effects / Q000378:metabolism*; D009435:Synaptic Transmission / Q000187:drug effects / Q000502:physiology; D016708:Synaptophysin / Q000378:metabolism; D013997:Time Factors; D015854:Up-Regulation / Q000187:drug effects / Q000502:physiology; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*
D018756:GABA Antagonists; D058787:GABA-A Receptor Antagonists; D011963:Receptors, GABA-A; D016708:Synaptophysin; D005680:gamma-Aminobutyric Acid; D005968:Glutamate Decarboxylase; D002118:Calcium