Gamma aminobutyric acid (GABA) has been established as
an important developmental signal in a number of regions
of the central nervous system (CNS), including retina.
Our previous studies have shown that GABAergic horizontal
cells act as the initial synaptic target for developing
cone photoreceptors in neonatal rabbit retina. Since intraocular
injections of the GABAA receptor antagonists,
picrotoxin or bicuculline, disrupt cone synaptogenesis
in vivo, GABA released from horizontal cells may
provide a necessary signal for cone axon growth and/or
synapse formation. In the current report, we have used
cultured retinal explants to examine the effects of GABAA
receptor antagonists on other aspects of developing cones.
These include the distribution pattern of cone cell bodies
across the outer surface of the retina and the expression
of GABAA receptors within both cone cell bodies
and axonal processes. Peanut agglutinin (PNA), a plant
lectin that specifically labels cone plasma membrane and
extracellular matrix, was used to monitor cone development,
and a GABAA receptor antibody against the β2/3
subunits of the protein was used to label GABAA
receptors. Results showed that cones maintained in the
explant culture express GABAA receptors in a
temporal and spatial pattern similar to that observed in
vivo, namely a low expression of receptors on cone
cell bodies at postnatal day 1 (P1), peaking around P3
and diminishing by P7. Neonatal retinal explants exposed
to the GABAA receptor antagonists, bicuculline
(10 μM) or SR95531 (5 μM), for 24 h in culture
showed disruption of the normal distribution of cone cell
bodies. When GABA (100 μM) was added along with either
antagonist, cone cell bodies appeared normal. Neither bicuculline
nor SR95531 alone had any effect on the general morphology
of other retinal layers, suggesting that these GABAA
receptor antagonists at the concentrations used were not
acting as nonspecific disruption agents. The effects of
GABA antagonists were confined to the first week after
birth with no disruption seen in P9 or adult explant cultures.
These data provide a direct demonstration of the necessity
for GABAergic input to cones during active synaptogenesis.
As we have previously shown, GABAA receptor
activation causes a substantial increase in intracellular
calcium concentrations in cones and thereby could provide
a mechanism by which GABA regulates cone maturation.