GABA uptake and release in purified neuronal and nonneuronal cultures from chick embryo retina

Abstract

Uptake and release of gamma-aminobutyric acid (GABA) have been studied using glia-free, purified neuronal cultures from 8-day chick embryo retina. At 3 days in vitro 65% of the neurons showed high-affinity GABA uptake. These neurons appeared heavily labeled after incubation in 5 X 10(-8) M [3H]GABA, but no labeling was detected when the incubation was carried out at 4 degrees C, or in the absence of Na+ ions. Diaminobutyric acid (DABA) also blocked completely the neuronal uptake of GABA, while beta -alanine was ineffective at similar concentrations. At 6 days in vitro Na+- and temperature-dependent GABA uptake was present in 50% of the neurons. In addition, in 80% of those neurons the uptake was insensitive to DABA or beta -alanine, whereas in the remaining 20% it was blocked by DABA but not by beta -alanine. Important developmental changes were also found in the capacity of the neurons to release GABA into the medium. Spontaneous GABA release (i.e. that taking place in regular medium, containing 5 mM K+) was higher at 3 than at 6 days in vitro. However, increasing the K+ concentration to 56 mM had minimal effects at 3 days in vitro, but induced a 2 to 3-fold increase in GABA release at 6 days in vitro. This K+-induced release appeared to be Ca2+-dependent, since it was substantially reduced the presence of 10 mM Co2+. Cultures containing a confluent monolayer of nonneuronal flat cells were generated by seeding retinal cell suspensions on poorly adhesive substrata. Retina nonneuronal cells showed, during the first 10 days in vitro, a high-affinity mechanism for GABA uptake which was Na+- and temperature-dependent, and was reduced by 85% by DABA but was practically unaffected by beta-alanine. This uptake mechanism seemed to be lost towards the end of the second week in vitro, and could not be detected after 21 days culture.