bafilomycin-a1 and cesium-chloride

GABA release by dopaminergic amacrine (DA) cells of the mouse retina was detected by measuring Cl- currents generated by isolated perikarya in response to their own neurotransmitter. The possibility that the Cl- currents were caused by GABA release from synaptic endings that had survived the dissociation of the retina was ruled out by examining confocal Z series of the surface of dissociated tyrosine hydroxylase-positive perikarya after staining with antibodies to pre and postsynaptic markers. GABA release was caused by exocytosis because 1) the current events were transient on the millisecond time scale and thus resembled miniature synaptic currents; 2) they were abolished by treatment with a blocker of the vesicular proton pump, bafilomycin A1; and 3) their frequency was controlled by the intracellular Ca2+ concentration. Because DA cell perikarya do not contain presynaptic active zones, release was by necessity extrasynaptic. A range of depolarizing stimuli caused GABA exocytosis, showing that extrasynaptic release of GABA is controlled by DA cell electrical activity. With all modalities of stimulation, including long-lasting square pulses, segments of pacemaker activity delivered by the action-potential-clamp method and high-frequency trains of ramps, discharge of GABAergic currents exhibited considerable variability in latency and duration, suggesting that coupling between Ca2+ influx and transmitter exocytosis is extremely loose in comparison with the synapse. Paracrine signaling based on extrasynaptic release of GABA by DA cells and other GABAergic amacrines may participate in controlling the excitability of the neuronal processes that interact synaptically in the inner plexiform layer.

Topics: Animals; Cadmium; Calcium; Cells, Cultured; Cesium; Chlorides; Dopamine; Dose-Response Relationship, Drug; Electric Stimulation; Enzyme Inhibitors; Exocytosis; GABA Antagonists; gamma-Aminobutyric Acid; Macrolides; Membrane Potentials; Mice; Mice, Inbred C57BL; Models, Biological; Neurons; Patch-Clamp Techniques; Pyridazines; Retina; Sodium Channel Blockers; Synapses; Synapsins; Tetrodotoxin; Tyrosine 3-Monooxygenase

Glutamate transporters are expressed throughout the CNS where their major role is to clear released glutamate from presynaptic terminals. Here, we report a novel function of the transporter in rat pinealocytes. This electrogenic transporter conducted inward current in response to L-glutamate and L- or D-aspartate and depolarized the membrane in patch-clamp experiments. Ca2+ imaging demonstrated that the transporter-mediated depolarization induced a significant Ca2+ influx through voltage-gated Ca2+ channels. The Ca2+ rise finally evoked glutamate exocytosis as detected by carbon-fiber amperometry and by HPLC. In pineal slices with densely packed pinealocytes, glutamate released from the cells effectively activated glutamate transporters in neighboring cells. The Ca2+ signal generated by KCl depolarization or acetylcholine propagated through several cell layers by virtue of the regenerative "glutamate-induced glutamate release." Therefore, we suggest that glutamate transporters mediate synchronized elevation of L-glutamate and thereby efficiently downregulate melatonin secretion via previously identified inhibitory metabotropic glutamate receptors in the pineal gland.

Topics: Amino Acid Transport System X-AG; Animals; Aspartic Acid; Calcium; Cells, Cultured; Cesium; Chlorides; Dose-Response Relationship, Drug; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Exocytosis; Glutamic Acid; In Vitro Techniques; Kainic Acid; Macrolides; Magnesium; Male; Melatonin; Membrane Potentials; Pineal Gland; Potassium Chloride; Rats; Rats, Sprague-Dawley