GABA and Glycine Activated Currents in the Rod Bipolar Cell of the Rabbit
Retina.
Gillette, Michael A., and Ramon F. Dacheux.
Department of Neurobiology, Harvard Medical School, Boston, Massachusetts
02115 and Department of Ophthalmology, University of Alabama at Birmingham,
Birmingham, AL 35233.
APStracts 2:0106N, 1995.
SUMMARY AND CONCLUSIONS
1. Voltage- and ligand-gated currents were recorded from solitary rabbit rod
bipolar cells using the whole-cell patch-clamp technique. The rod bipolar
forms a single, stereotypical physiological and morphological class of cell
which was easily identified from other neurons and support cells after
enzymatic and mechanical dissociation from isolated retina. Protein kinase C
immunoreactivity confirmed the validity of using a purely morphological
identification of this cell type. 2. Voltage steps of 15 mV increments from a
holding potential of -45 mV elicited a large outward current activated near -
30 mV. These voltage-gated currents were eliminated by using equimolar
substitutions of Cs+ and TEA+ for K+ in the pipet, indicating that they
represent a mixture of K+ currents. 3. The putative inhibitory
neurotransmitters, g-aminobutyric acid (GABA) and glycine, activated inward
Cl- currents when pressure-applied from pipets placed near the axon terminals
of rod bipolar cell which were voltage clamped at -45 mV. With changes in
intracellular or extracellular Cl- concentration, the reversal potential of
these ligand gated currents changed as predicted by the Nernst equation for
Cl- activity. The dose-response curves for GABA and glycine were sigmoidal
with saturating concentrations of 100 mM and 300 mM, respectively. 4. GABA-
activated currents were: 1) reversibly reduced by the allosteric inhibitor,
picrotoxin, and the competitive antagonist, bicuculline; 2) potentiated by the
benzodiazepine, Diazepam, and the barbiturate, sodium barbital; and 3)
indistinguishable from muscimol-activated currents. There was no response to
the GABAB agonist, baclofen. Collectively, these data strongly suggested that
the GABA-activated currents in rabbit rod bipolar cells were mediated by the
GABAA receptor. This was similar to the GABA-activated currents in other
mammalian rod bipolar cells. 5. Application of the conformationally restricted
GABA analogue cis-4-aminocrotonic acid, CACA, failed to elicit a response,
while the conformationally extended GABA analogue trans-4-aminocrotonic acid,
TACA, elicited a response similar to that of GABA. Though bicuculline appeared
to suppress the GABA-activated current slightly more than the TACA-activated
one (not significant using Student's t-distribution), GABA- and TACA-activated
currents were equally suppressed by picrotoxin and equally enhanced by
Diazepam and sodium barbital. These data coupled with the inefficacy of CACA
argued against the existence of a GABAC-type channel in the rod bipolar cell
of the rabbit, and suggested that GABA and TACA were activating the same GABAA
receptor-channel complex. 6. The rabbit rod bipolar cell, like those of other
mammalian retinas, demonstrated the presence of conventional glycine receptors
which were antagonized by nanomolar and low micromolar concentrations of
strychnine. 7. Focal application of GABA and glycine was used to examine the
regional distribution of chemical sensitivity across the cell. Ligand-
activated currents elicited from the rod bipolar cell terminals were always
larger than those generated from the soma. To correlate the distribution of
the sensitivity with a visual representation of a receptor density, solitary
rod bipolar cells where incubated with BODIPY-labeled receptor specific
neuroactive substances. BODIPY-Ro-1986, a benzodazepine receptor ligand, used
to visualize the GABAA receptors demonstrated no discernible focality of
receptor distribution, a result incompatible with the electrophysiological
data. By contrast, glycine receptors labeled with BODIPY-strychnine, the
glycine antagonist, demonstrated the highest density of receptors on the axon
terminal, intermediate receptor density on the soma, and virtually no labeling
on the dendrites, a result consistent with predictions based upon
electrophysiology. 8. Dopamine, 5-hydroxytryptamine, and adenosine were
applied either individually to evaluate direct current responses or in
conjunction with GABA and glycine to determine their ability to modulate the
known ligand-gated currents. None of these putative transmitters, shown by
immunocytochemistry to be localized to the region of the IPL were rabbit rod
bipolar cell terminals reside, either activated a current or modulated the
GABA- or glycine-activated currents. 9. The neuroactive peptides substance P,
substance Y, somatostatin, neurotensin and vasoactive intestinal peptide
(VIP), have also been localized by immunocytochemical studies to the strata of
the IPL in which the rod bipolar axon endings terminate. When these substances
were applied singly to the rod bipolar cell during whole cell recording
experiments, they elicited no observable current response at any holding
potential. However, the application of a prepulse of VIP prior to the test
pulse of GABA reduced the GABA-activated current by an average of 37% in the
45 cells tested; a similar prepulse had no affect on the glycine-activated
currents. The remaining neuroactive peptides had no modulatory effect on GABA-
and glycine-activated currents.
Received 2 December 1994; accepted in final form 29 March 1995.
APS Manuscript Number J754-4.
Article publication pending J. Neurophysiol.
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 1 May 1995.