Feedback from horizontal cells (HCs) to cone photoreceptors plays a key role in the center-surround–receptive field organization of retinal neurons. Recordings from cone photoreceptors in newt retinal slices were obtained by the whole-cell patch-clamp technique, using a superfusate containing a GABA antagonist (100 μM picrotoxin). Surround illumination of the receptive field increased the voltage-dependent calcium current (ICa) in the cones, and shifted the activation voltage of ICa to negative voltages. External alkalinization also increased cone ICa and shifted its activation voltage toward negative voltages. Enrichment of the pH buffering capacity of the extracellular solution increased cone ICa, and blocked any additional increase in cone ICa by surround illumination. Hyperpolarization of the HCs by a glutamate receptor antagonist-augmented cone ICa, whereas depolarization of the HCs by kainate suppressed cone ICa. From these results, we propose the hypothesis that pH changes in the synaptic clefts, which are intimately related to the membrane voltage of the HCs, mediate the feedback from the HCs to cone photoreceptors. The feedback mediated by pH changes in the synaptic cleft may serve as an additional mechanism for the center-surround organization of the receptive field in the outer retina.

Numerous neurons release two transmitters of low molecular mass, but it is controversial whether they are localized within the same synaptic vesicle, with the single exception of GABA and glycine because they are ferried into the vesicle by the same transporter. Retinal dopaminergic amacrine (DA) cells synthesize both dopamine and GABA. Both transmitters are released over the entire cell surface and act on neighboring and distant neurons by volume transmission, but, in addition, DA cells establish GABAergic synapses onto AII amacrine cells, the neurons that transfer rod signals to cone bipolars. By combining recordings of dopamine and GABA release from isolated, genetically identified perikarya of DA cells from the mouse retina, we observed that a proportion of the events of dopamine and GABA exocytosis were simultaneous, suggesting co-release. Furthermore, a proportion of the secretory organelles in the perikaryon and synaptic endings of DA cells contained both vesicular transporters for dopamine (VMAT2) and GABA (VGAT). Since the majority of the dopamine release events concerned a single transmitter and organelles were present that contained a single transporter, either VMAT2 or VGAT, we conclude that the secretory organelles of DA cells contain variable concentrations of the two transmitters, which are in turn determined by a variable mixture of the two transporter molecules in their limiting membrane. This variability can be explained if the relative numbers of transporter molecules is determined stochastically during the budding of the somatic organelles from the trans-Golgi-network or the retrieval of the vesicular membrane from the plasmalemma after exocytosis.

Hirasawa H, Puopolo M, Raviola E. Extrasynaptic release of GABA by retinal dopaminergic neurons. J Neurophysiol 102: 146 -158, 2009. First published April 29, 2009 doi:10.1152/jn.00130.2009. GABA release by dopaminergic amacrine (DA) cells of the mouse retina was detected by measuring Cl Ϫ currents generated by isolated perikarya in response to their own neurotransmitter. The possibility that the Cl Ϫ 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 hydroxylasepositive perikarya after staining with antibodies to preand 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 Ca 2ϩ 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 actionpotential-clamp method and high-frequency trains of ramps, discharge of GABAergic currents exhibited considerable variability in latency and duration, suggesting that coupling between Ca 2ϩ 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.

The mammalian neural retina contains single or multiple intrinsic circadian oscillators that can be directly entrained by light cycles. Dopaminergic amacrine (DA) cells represent an especially interesting candidate as a site of the retinal oscillator because of the crucial role of dopamine in light adaptation, and the widespread distribution of dopamine receptors in the retina. We hereby show by single-cell, end-point RT-PCR that retinal DA cells contain the transcripts for six core components of the circadian clock: Bmal1, Clock, Cry1, Cry2, Per1, and Per2. Rod photoreceptors represented a negative control, because they did not appear to contain clock transcripts. We finally confirmed that DA cells contain the protein encoded by the Bmal1 gene by comparing immunostaining of the nuclei of DA cells in the retinas of wildtype and Bmal1-/- mice. It is therefore likely that DA cells contain a circadian clock that anticipates predictable variations in retinal illumination.

One contribution of 16 to a discussion meeting issue 'Release of chemical transmitters from cell bodies and dendrites of nerve cells'. In the mouse retina, dopaminergic amacrine (DA) cells synthesize both dopamine and GABA. Both transmitters are released extrasynaptically and act on neighbouring and distant retinal neurons by volume transmission. In simultaneous recordings of dopamine and GABA release from isolated perikarya of DA cells, a proportion of the events of dopamine and GABA exocytosis were simultaneous, suggesting co-release. In addition, DA cells establish GABAergic synapses onto AII amacrine cells, the neurons that transfer rod bipolar signals to cone bipolars. GABA A but not dopamine receptors are clustered in the postsynaptic membrane. Therefore, dopamine, irrespective of its site of release-synaptic or extrasynaptic-exclusively acts by volume transmission. Dopamine is released upon illumination and sets the gain of retinal neurons for vision in bright light. The GABA released at DA cells' synapses probably prevents signals from the saturated rods from entering the cone pathway when the dark-adapted retina is exposed to bright illumination. The GABA released extrasynaptically by DA and other amacrine cells may set a 'GABAergic tone' in the inner plexiform layer and thus counteract the effects of a spillover of glutamate released at the bipolar cell synapses of adjacent OFF and ON strata, thus preserving segregation of signals between ON and OFF pathways.