Previously studied for its role in processing olfactory information in the antennal lobe, GABA also may shape development of the olfactory pathway, acting either through or on glial cells. Early in development, the dendrites of GABAergic neurons extend to the glial border that surrounds the nascent olfactory lobe neuropil. These neuropil glia express both GABAA and GABAB receptors, about half of the glia in acute cultures responded to GABA with small outward currents, and about a third responded with small transient increases in intracellular calcium. The neuronal classes that express GABA in vivo, the local interneurons and a subset of projection neurons, also do so in culture. Exposure to GABA in culture increased the size and complexity of local interneurons, but had no effect on glial morphology. The presence of glia alone did not affect neuronal morphology, but in the presence of both glia and GABA, the growth-enhancing effects of GABA on cultured antennal lobe neurons were eliminated. Contact between the glial cells and the neurons was not necessary. Operating in vivo, these antagonistic effects, one direct and one glia mediated, could help to sculpt the densely branched, tufted arbors that are characteristic of neurons innervating olfactory glomeruli.

The somatic muscle cells of the parasitic nematode Ascaris suum possess GABA receptors that gate chloride conductances in a similar fashion to the mammalian GABAA receptor subtype. These receptors mediate muscle relaxation and are the site of action of the anthelmintic piperazine. The properties of this receptor differ from the properties of the GABA-gated chloride receptors in the mammalian host, in particular they are not as sensitive to mammalian GABA receptor antagonists such as bicuculline and picrotoxin. Using two-electrode intracellular electrophysiological recording techniques from Ascaris muscle cells, we have tested the potency of a series of azole derivatives for their ability to block the chloride-dependent GABA response. The lead compound, SN606078, 2-(2,6-dichloro-4-trifluromethylphenyl)-4-(4,5-dicyano-lH-imidazol-2-yl)-2H-l,2,3-triazoIe, and 4 structurally related compounds reversibly blocked the conductance increase elicited by 30 μM GABA with IC50s of less than 10 μM. SN606078 (10 μM) decreased the slope of the dose-response curve for GABA, suggesting a non-competitive mechanism of action. In two-electrode voltage clamp experiments, 10μM SN606078 blocked the outward current elicited by 20 μM GABA in a voltage-dependent manner with 72 ± 2% inhibition at −20 mV and 49±6% inhibition at −40 mV. These observations indicate that SN606078 may act as an open-channel blocker of the GABA-gated chloride channel in A. suum.

Vertical Slices of postnatal day 6 (P6) rat retina were cut and cultured using the roller-tube technique. The organotypic differentiation during a culture period of up to 30 days has been described in a previous study (Feigenspan et al., 1993a). Here we concentrated on the synaptic organization in the retinal slice culture. Electron microscopy revealed the presence of ribbon synapses in the outer plexiform layer and conventional and ribbon syanpses in the inner plexiform layer. Immunofluroscence with antibodies that recognize specific subunits of GABAA or glycine receptors revealed a punctuate distribution of the receptors. They were aggregated in “hot spots” that correspond to a concentration of receptors at postsynaptic sites. Different isoforms of GABAA and glycine receptors occured in the slice cultures. The experiments show that there is a differentiation of synapses and a diversity of transmitter receptors in the slice cultures that is comparable to the in vivo retina.

The nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) signal transduction pathway plays a role in every retinal cell type. Previous studies have shown that excitatory glutamatergic synaptic pathways can increase cGMP-like immunoreactivity (cGMP-LI) in retina through stimulation of NO production, but little is known about the role of synaptic inhibition in the modulation of cGMP-LI. Gamma-amino-n-butyric acid (GABA) plays critical roles in modulating excitatory synaptic pathways in the retina. Therefore, we used GABA receptor antagonists to explore the role of GABAergic inhibitory synaptic pathways on the modulation of the NO/cGMP signal-transduction system. Cyclic GMP immunocytochemistry was used to investigate the effects of the GABA receptor antagonists bicuculline, picrotoxin, and (1,2,5,6-tetrahyropyridin-4-yl) methylphosphinic acid (TPMPA) on levels of cGMP-LI. Cyclic GMP-LI was strongly increased in response to the GABAA receptor antagonist bicuculline, while the GABAC receptor antagonist TPMPA had little effect on cGMP-LI. The GABAA/GABAC receptor antagonist, picrotoxin, caused a moderate increase in cGMP-LI, which was mimicked by the combination of bicuculline and TPMPA. The nitric oxide synthase inhibitor, S-methyl-L-thiocitrulline (SMTC), blocked the increased cGMP-LI in response to stimulation with either bicuculline or picrotoxin. Treatments with either of the glutamate receptor antagonists (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801) or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) partially blocked the increases in cGMP-LI seen in response to bicuculline, but a combination of MK-801 and CNQX completely eliminated these increases. These results suggest that inhibitory synaptic pathways involving both types of GABA receptors work through excitatory glutamatergic receptors to regulate the NO/cGMP signal-transduction pathway in retina.

The major inhibitory neurotransmitters GABA and glycine provide the bulk of input to large-field ganglion cells in the retina. Whole-cell patch-clamp recordings were used to characterize the glycine- and GABA-activated currents for morphologically identified ON-α ganglion cells in the rabbit retina. Cells identified as ON-α cells by light evoked currents were intracellularly stained and examined by light microscopy which revealed dendritic stratification in the vitreal half of the inner plexiform layer and confirmed their physiological identity. All Ca2+-mediated synaptic influences were abolished with Co2+, revealing two types of ON-α cell characterized by their different inhibitory current profiles. One group exhibited larger glycine- than GABA-activated currents, while the other group had larger GABA- than glycine-activated currents. Both cell types demonstrated strychnine-sensitive glycine-activated currents and bicuculline-sensitive GABAA-activated currents. Surprisingly, both cell types expressed functional GABAC receptors demonstrated by their sensitivity to TPMPA. In addition, the cells with larger glycine-activated currents also possessed GABAB receptors, whereas those with larger GABA-activated currents did not. Immunocytochemical experiments confirmed the presence of glycine, GABAA, and GABAC receptor subunits on all physiologically identified ON-α ganglion cells in this study. In addition, the GABAB receptor immunolabeled puncta were present on the cells with larger glycine-activated currents, but not on the cells with the larger GABA-activated currents. In conclusion, the presence of different functional GABA and glycine receptors determined physiologically correlated well with the specific GABA and glycine receptor immunolabeling for two neuropharmacological types of rabbit ON-α ganglion cells.

Inhibitory synaptic transmission via GABA and glycine receptors plays a crucial role in shaping the excitatory response of neurons in the retina. Whole-cell recordings were obtained from ganglion cells in the intact rabbit eyecup preparation to correlate GABA- and glycine-activated currents with the presence of their specific receptors on morphologically identified α ganglion cells. Alpha ganglion cells were chosen based upon their large somata when viewing the retinal surface, and responses to light and dark spots were used to identify OFF-alpha ganglion cells. Light responses were abolished by superfusion of Ringer's containing cobalt to synaptically isolate the cell by blocking all Ca2+-mediated transmitter release. Pressure pulses of GABA and glycine were delivered to an area that encompassed the dendritic field while receptor antagonists were applied through superfusion to characterize the direct inhibition onto the ganglion cell. Physiological results indicated that OFF-α cells did not have any GABAC receptor-activated currents, but did express currents mediated by ionotropic GABAA receptors and metabotropic GABAB receptors that were blocked by their specific antagonists bicuculline and CGP55845, respectively. The amplitudes of strychnine-sensitive glycine-activated currents were always larger than the currents elicited by GABA. Confocal optical sections of physiologically identified, sulforhodamine B-stained cells displayed the localization of glycine and GABAA receptor subunit labeling dispersed over the stained dendrites.