Reductions in Calcium Signaling Limit Inhibition to Diabetic Retinal Rod Bipolar Cells.

Purpose: The balance of neuronal excitation and inhibition is important for proper retinal signaling. A previous report showed that diabetes selectively reduces light-evoked inhibition to the retinal dim light rod pathway, changing this balance. Here, changes in mechanisms of retinal inhibitory synaptic transmission after 6 weeks of diabetes are investigated. Methods: Diabetes was induced in C57BL/6J mice by three intraperitoneal injections of streptozotocin (STZ, 75 mg/kg), and confirmed by blood glucose levels more than 200 mg/dL. After 6 weeks, whole-cell voltage-clamp recordings of electrically evoked inhibitory postsynaptic currents from rod bipolar cells and light-evoked excitatory postsynaptic currents from A17-amacrine cells were made in dark-adapted retinal slices. Results: Diabetes shortened the timecourse of directly activated lateral GABAergic inhibitory amacrine cell inputs to rod bipolar cells. The timing of GABA release onto rod bipolar cells depends on a prolonged amacrine cell calcium signal that is reduced by slow calcium buffering. Therefore, the effects of calcium buffering with EGTA-acetoxymethyl ester (AM) on diabetic GABAergic signaling were tested. EGTA-AM reduced GABAergic signaling in diabetic retinas more strongly, suggesting that diabetic amacrine cells have reduced calcium signals. Additionally, the timing of release from reciprocal inhibitory inputs to diabetic rod bipolar cells was reduced, but the activation of the A17 amacrine cells responsible for this inhibition was not changed. Conclusions: These results suggest that reduced light-evoked inhibitory input to rod bipolar cells is due to reduced and shortened calcium signals in presynaptic GABAergic amacrine cells. A reduction in calcium signaling may be a common mechanism limiting inhibition in the retina.

Dopamine D1 receptor activation contributes to light-adapted changes in retinal inhibition to rod bipolar cells.

Dopamine modulation of retinal signaling has been shown to be an important part of retinal adaptation to increased background light levels, but the role of dopamine modulation of retinal inhibition is not clear. We previously showed that light adaptation causes a large reduction in inhibition to rod bipolar cells, potentially to match the decrease in excitation after rod saturation. In this study, we determined how dopamine D1 receptors in the inner retina contribute to this modulation. We found that D1 receptor activation significantly decreased the magnitude of inhibitory light responses from rod bipolar cells, whereas D1 receptor blockade during light adaptation partially prevented this decline. To determine what mechanisms were involved in the modulation of inhibitory light responses, we measured the effect of D1 receptor activation on spontaneous currents and currents evoked from electrically stimulating amacrine cell inputs to rod bipolar cells. D1 receptor activation decreased the frequency of spontaneous inhibition with no change in event amplitudes, suggesting a presynaptic change in amacrine cell activity in agreement with previous reports that rod bipolar cells lack D1 receptors. Additionally, we found that D1 receptor activation reduced the amplitude of electrically evoked responses, showing that D1 receptors can modulate amacrine cells directly. Our results suggest that D1 receptor activation can replicate a large portion but not all of the effects of light adaptation, likely by modulating release from amacrine cells onto rod bipolar cells. NEW & NOTEWORTHY We demonstrated a new aspect of dopaminergic signaling that is involved in mediating light adaptation of retinal inhibition. This D1 receptor-dependent mechanism likely acts through receptors located directly on amacrine cells, in addition to its potential role in modulating the strength of serial inhibition between amacrine cells. Our results also suggest that another D2/D4 receptor-dependent or dopamine-independent mechanism must also be involved in light adaptation of inhibition to rod bipolar cells.

Early Retinal Neuronal Dysfunction in Diabetic Mice: Reduced Light-Evoked Inhibition Increases Rod Pathway Signaling.

PURPOSE: Recent studies suggest that the neural retinal response to light is compromised in diabetes. Electroretinogram studies suggest that the dim light retinal rod pathway is especially susceptible to diabetic damage. The purpose of this study was to determine whether diabetes alters rod pathway signaling. METHODS: Diabetes was induced in C57BL/6J mice by three intraperitoneal injections of streptozotocin (STZ; 75 mg/kg), and confirmed by blood glucose levels > 200 mg/dL. Six weeks after the first injection, whole-cell voltage clamp recordings of spontaneous and light-evoked inhibitory postsynaptic currents from rod bipolar cells were made in dark-adapted retinal slices. Light-evoked excitatory currents from rod bipolar and AII amacrine cells, and spontaneous excitatory currents from AII amacrine cells were also measured. Receptor inputs were pharmacologically isolated. Immunohistochemistry was performed on whole mounted retinas. RESULTS: Rod bipolar cells had reduced light-evoked inhibitory input from amacrine cells but no change in excitatory input from rod photoreceptors. Reduced light-evoked inhibition, mediated by both GABAA and GABAC receptors, increased rod bipolar cell output onto AII amacrine cells. Spontaneous release of GABA onto rod bipolar cells was increased, which may limit GABA availability for light-evoked release. These physiological changes occurred in the absence of retinal cell loss or changes in GABAA receptor expression levels. CONCLUSIONS: Our results indicate that early diabetes causes deficits in the rod pathway leading to decreased light-evoked rod bipolar cell inhibition and increased rod pathway output that provide a basis for the development of early diabetic visual deficits.

Different types of retinal inhibition have distinct neurotransmitter release properties.

Neurotransmitter release varies between neurons due to differences in presynaptic mechanisms such as Ca(2+) sensitivity and timing. Retinal rod bipolar cells respond to brief dim illumination with prolonged glutamate release that is tuned by the differential release of GABA and glycine from amacrine cells in the inner retina. To test if differences among types of GABA and glycine release are due to inherent amacrine cell release properties, we directly activated amacrine cell neurotransmitter release by electrical stimulation. We found that the timing of electrically evoked inhibitory currents was inherently slow and that the timecourse of inhibition from slowest to fastest was GABAC receptors > glycine receptors > GABAA receptors. Deconvolution analysis showed that the distinct timing was due to differences in prolonged GABA and glycine release from amacrine cells. The timecourses of slow glycine release and GABA release onto GABAC receptors were reduced by Ca(2+) buffering with EGTA-AM and BAPTA-AM, but faster GABA release on GABAA receptors was not, suggesting that release onto GABAA receptors is tightly coupled to Ca(2+). The differential timing of GABA release was detected from spiking amacrine cells and not nonspiking A17 amacrine cells that form a reciprocal synapse with rod bipolar cells. Our results indicate that release from amacrine cells is inherently asynchronous and that the source of nonreciprocal rod bipolar cell inhibition differs between GABA receptors. The slow, differential timecourse of inhibition may be a mechanism to match the prolonged rod bipolar cell glutamate release and provide a way to temporally tune information across retinal pathways.

Slow changes in Ca2(+) cause prolonged release from GABAergic retinal amacrine cells.

The timing of neurotransmitter release from neurons can be modulated by many presynaptic mechanisms. The retina uses synaptic ribbons to mediate slow graded glutamate release from bipolar cells that carry photoreceptor inputs. However, many inhibitory amacrine cells, which modulate bipolar cell output, spike and do not have ribbons for graded release. Despite this, slow glutamate release from bipolar cells is modulated by slow GABAergic inputs that shorten the output of bipolar cells, changing the timing of visual signaling. The time course of light-evoked inhibition is slow due to a combination of receptor properties and prolonged neurotransmitter release. However, the light-evoked release of GABA requires activation of neurons upstream from the amacrine cells, so it is possible that prolonged release is due to slow amacrine cell activation, rather than slow inherent release properties of the amacrine cells. To test this idea, we directly activated primarily action potential-dependent amacrine cell inputs to bipolar cells with electrical stimulation. We found that the decay of GABAC receptor-mediated electrically evoked inhibitory currents was significantly longer than would be predicted by GABAC receptor kinetics, and GABA release, estimated by deconvolution analysis, was inherently slow. Release became more transient after increasing slow Ca(2+) buffering or blocking prolonged L-type Ca(2+) channels and Ca(2+) release from intracellular stores. Our results suggest that GABAergic amacrine cells have a prolonged buildup of Ca(2+) in their terminals that causes slow, asynchronous release. This could be a mechanism of matching the time course of amacrine cell inhibition to bipolar cell glutamate release.

Upregulation of synaptotagmin IV inhibits transmitter release in PC12 cells with targeted synaptotagmin I knockdown.

BACKGROUND: The function of synaptotagmins (syt) in Ca2+-dependent transmitter release has been attributed primarily to Ca2+-dependent isoforms such as syt I. Recently, syt IV, an inducible Ca2+-independent isoform has been implicated in transmitter release. We postulated that the effects of syt IV on transmitter release are dependent on the expression of syt I. RESULTS: To test this, we increased syt IV expression in PC12 cells by either upregulation with forskolin treatment or overexpression with transfection. Two separately generated stable PC12 cell lines with syt I expression abolished by RNAi targeting were used and compared to control cells. We measured catecholamine release from single vesicles by amperometry and neuropeptide Y release from populations of cells by an immunoassay. In syt I targeted cells with forskolin-induced syt IV upregulation, amperometry measurements showed a reduction in the number of release events and the total amount of transmitter molecules released per cell. In cells with syt IV overexpressed, similar amperometry results were obtained, except that the rate of expansion for full fusion was slowed. Neuropeptide Y (NPY) release from syt I knockdown cells was decreased, and overexpression of syt IV did not rescue this effect. CONCLUSIONS: These data support an inhibitory effect of syt IV on release of vesicles and their transmitter content. The effect became more pronounced when syt I expression was abolished.