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Although diabetic retinopathy (DR) is clinically diagnosed as a vascular disease, many studies find retinal neuronal and visual dysfunction before the onset of vascular DR. This suggests that DR should be viewed as a neurovascular disease. Prior to the onset of DR, human patients have compromised electroretinograms that indicate a disruption of normal function, particularly in the inner retina. They also exhibit reduced contrast sensitivity. These early changes, especially those due to dysfunction in the inner retina, are also seen in rodent models of diabetes in the early stages of the disease. Rodent models of diabetes exhibit several neuronal mechanisms, such as reduced evoked GABA release, increased excitatory glutamate signaling, and reduced dopamine signaling, that suggest specific neuronal deficits. This suggests that understanding neuronal deficits may lead to early diabetes treatments to ameliorate neuronal dysfunction. Expected final online publication date for the Annual Review of Vision Science, Volume 9 is September 2023. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

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.

Purpose: It has been known for some time that normal retinal signaling is disrupted early on in diabetes, before the onset of the vascular pathologies associated with diabetic retinopathy. There is growing evidence that levels of retinal dopamine, a neuromodulator that mediates light adaptation, may also be reduced in early diabetes. Previously, we have shown that after six weeks of diabetes in a mouse model, light adaptation is impaired at the level of ON-sustained (ON-s) ganglion cells. The purpose of this study was to determine whether changes in dopamine receptor sensitivity contribute to this dysfunction. Here we used single cell retinal patch-clamp recordings from the mouse retina to determine how activating dopamine type D4 receptors (D4Rs) changes the light-evoked and spontaneous excitatory inputs to ON-s ganglion cells, in both control and diabetic animals. We also used in-situ fluorescent hybridization to assess whether D4R expression was impacted by diabetes. We found that D4R activation had a smaller impact on light-evoked excitatory inputs to ON-s ganglion cells in diabetic retinas compared to controls. This impaired D4R signaling is not attributable to a decline in D4R expression, as we found increased D4R mRNA density in the outer plexiform layer in diabetic retinas. This suggests that the cellular machinery of dopaminergic signaling is itself disrupted in early diabetes and may be amenable to chronic dopamine supplementation therapy.

The adaptation of ganglion cells to increasing light levels is a crucial property of the retina. The retina must respond to light intensities that vary by 10-12 orders of magnitude, but the dynamic range of ganglion cell responses covers only ∼3 orders of magnitude. Dopamine is a crucial neuromodulator for light adaptation and activates receptors in the D1 and D2 families. D1Rs are expressed on horizontal cells and some bipolar, amacrine and ganglion cells. In the D2 family D2Rs are expressed on dopaminergic amacrine cells and D4Rs are primarily expressed on photoreceptors. However, the roles of activating these receptors to modulate the synaptic properties of the inputs to ganglion cells are not yet clear. Here we used single cell retinal patch-clamp recordings from the mouse retina to determine how activating D1Rs and D4Rs changed the light-evoked and spontaneous excitatory inputs to ON-sustained (ON-s) ganglion cells. We found that both D1R and D4R activation decrease the light-evoked excitatory inputs to ON-s ganglion cells, but that only the sum of the peak response decrease due to activating the two receptors was similar to the effect of light adaptation to a rod-saturating background. The largest effects on spontaneous excitatory activity of both D1R and D4R agonists was on the frequency of events, suggesting that both D1Rs and D4Rs are acting upstream of the ganglion cells.New and NoteworthyDopamine by bright light conditions allows retinal neurons to reduce sensitivity to adapt to bright light conditions. It is not clear how and why dopamine receptors modulate retinal ganglion cell signaling. We found that both D1 and D4 dopamine receptors in photoreceptors and inner retinal neurons contribute significantly to the reduction in sensitivity of ganglion cells with light adaptation. However, light adaptation also requires dopamine independent mechanisms that could reflect inherent sensitivity changes in photoreceptors.

Retinal signaling under dark-adapted conditions is perturbed during early diabetes. Additionally, dopamine, the main neuromodulator of retinal light adaptation, is diminished in diabetic retinas. However, it is not known if this dopamine deficiency changes how the retina responds to increased light or dopamine. Here we determine whether light adaptation is impaired in the diabetic retina, and investigate potential mechanism(s) of impairment. Diabetes was induced in C57BL/6J male mice via 3 intraperitoneal injections of streptozotocin (75 mg/kg) and confirmed by blood glucose levels more than 200 mg/dL. After 6 weeks, whole-cell recordings of light-evoked and spontaneous inhibitory postsynaptic currents (IPSCs) or excitatory postsynaptic currents (EPSCs) were made from rod bipolar cells and ON sustained ganglion cells, respectively. Light responses were recorded before and after D1 receptor (D1R) activation (SKF-38393, 20 μM) or light adaptation (background of 950 photons·μm−2 ·s−1). Retinal whole mounts were stained for either tyrosine hydroxylase and activated caspase-3 or GAD65/67, GlyT1 and RBPMS and imaged. D1R activation and light adaptation both decreased inhibition, but the disinhibition was not different between control and diabetic rod bipolar cells. However, diabetic ganglion cell light-evoked EPSCs were increased in the dark and showed reduced light adaptation. No differences were found in light adaptation of spontaneous EPSC parameters, suggesting upstream changes. No changes in cell density were found for dopaminergic, glycinergic or GABAergic amacrine cells, or ganglion cells. Thus, in early diabetes, ON sustained ganglion cells receive excessive excitation under dark- and light-adapted conditions. Our results show that this is not attributable to loss in number or dopamine sensitivity of inhibitory amacrine cells or loss of dopaminergic amacrine cells.

During adaptation to an increase in environmental luminance, retinal signaling adjustments are mediated by the neuromodulator dopamine. Retinal dopamine is released with light and can affect center-surround receptive fields, the coupling state between neurons, and inhibitory pathways through inhibitory receptors and neurotransmitter release. While the inhibitory receptive field surround of bipolar cells becomes narrower and weaker during light adaptation, it is unknown how dopamine affects bipolar cell surrounds. If dopamine and light have similar effects, it would suggest that dopamine could be a mechanism for light-adapted changes. We tested the hypothesis that dopamine D1 receptor activation is sufficient to elicit the magnitude of light-adapted reductions in inhibitory bipolar cell surrounds. Surrounds were measured from OFF bipolar cells in dark-adapted mouse retinas while stimulating D1 receptors, which are located on bipolar, horizontal, and inhibitory amacrine cells. The D1 agonist SKF-38393 narrowed and weakened OFF bipolar cell inhibitory receptive fields but not to the same extent as with light adaptation. However, the receptive field surround reductions differed between the glycinergic and GABAergic components of the receptive field. GABAergic inhibitory strength was reduced only at the edges of the surround, while glycinergic inhibitory strength was reduced across the whole receptive field. These results expand the role of retinal dopamine to include modulation of bipolar cell receptive field surrounds. Additionally, our results suggest that D1 receptor pathways may be a mechanism for the light-adapted weakening of glycinergic surround inputs and the furthest wide-field GABAergic inputs to bipolar cells. However, remaining differences between light-adapted and D1 receptor–activated inhibition demonstrate that non-D1 receptor mechanisms are necessary to elicit the full effect of light adaptation on inhibitory surrounds.

Diabetic retinopathy is now well understood as a neurovascular disease. Significant deficits early in diabetes are found in the inner retina that consists of bipolar cells that receive inputs from rod and cone photoreceptors, ganglion cells that receive inputs from bipolar cells, and amacrine cells that modulate these connections. These functional deficits can be measuredin vivoin diabetic humans and animal models using the electroretinogram (ERG) and behavioral visual testing. Early effects of diabetes on both the human and animal model ERGs are changes to the oscillatory potentials that suggest dysfunctional communication between amacrine cells and bipolar cells as well as ERG measures that suggest ganglion cell dysfunction. These are coupled with changes in contrast sensitivity that suggest inner retinal changes. Mechanisticin vitroneuronal studies have suggested that these inner retinal changes are due to decreased inhibition in the retina, potentially due to decreased gamma aminobutyric acid (GABA) release, increased glutamate release, and increased excitation of retinal ganglion cells. Inner retinal deficits in dopamine levels have also been observed that can be reversed to limit inner retinal damage. Inner retinal targets present a promising new avenue for therapies for early-stage diabetic eye disease.

Purpose Retinal neuronal signaling is disrupted early in diabetes, before the onset of the vascular pathologies associated with diabetic retinopathy. There is also growing evidence that retinal dopamine, a neuromodulator that mediates light adaptation, is reduced in early diabetes. Previously, we have shown that after 6 weeks of diabetes, light adaptation is impaired in ON-sustained (ON-s) ganglion cells in the mouse retina. The purpose of this study was to determine whether changes in the response to dopamine receptor activation contribute to this dysfunction. Methods Single-cell retinal patch-clamp recordings from the mouse retina were used to determine how activating dopamine type D4 receptors (D4Rs) changes the light-evoked and spontaneous excitatory inputs to ON-s ganglion cells, in both control and 6-week diabetic (STZ-injected) animals. Fluorescence in situ hybridization was also used to assess whether D4R expression was affected by diabetes. Results D4R activation decreased light-evoked and spontaneous inputs to ON-s ganglion cells in control and diabetic retinas. However, D4R activation caused a smaller reduction in light-evoked excitatory inputs to ON-s ganglion cells in diabetic retinas compared to controls. This impaired D4R signaling is not attributable to a decline in D4R expression, as there was no change in D4R mRNA density in the diabetic retinas. Conclusions These results suggest that the cellular response to dopamine signaling is disrupted in early diabetes and may be amenable to chronic dopamine supplementation therapy.

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.

During adaptation from dim to bright environments, changes in retinal signaling are mediated, in part, by dopamine. Dopamine is released with light and can modulate retinal receptive fields, neuronal coupling, inhibitory receptors, and rod pathway inhibition. However, it is unclear how dopamine affects inner retinal inhibition to cone bipolar cells, which relay visual information from photoreceptors to ganglion cells and are important signal processing sites. We tested the hypothesis that dopamine (D)1 receptor activation is sufficient to elicit light-adapted inhibitory changes. Local light-evoked inhibition and spontaneous activity were measured from OFF cone bipolar cells in dark-adapted mouse retinas while stimulating D1 receptors, which are located on bipolar, horizontal, and inhibitory amacrine cells. The D1 agonist SKF38393 reduced local inhibitory light-evoked response magnitude and increased response transience, which mimicked changes measured with light adaptation. D1-mediated reductions in local inhibition were more pronounced for glycinergic than GABAergic inputs, comparable with light adaptation. The effects of D1 receptors on light-evoked input were similar to the effects on spontaneous input. D1 receptor activation primarily decreased glycinergic spontaneous current frequency, similar to light adaptation, suggesting mainly a presynaptic amacrine cell site of action. These results expand the role of dopamine to include signal modulation of cone bipolar cell local inhibition. In this role, D1 receptor activation, acting primarily through glycinergic amacrine cells, may be an important mechanism for the light-adapted reduction in OFF bipolar cell inhibition since the actions are similar and dopamine is released during light adaptation. NEW & NOTEWORTHY Retinal adaptation to different luminance conditions requires the adjustment of local circuits for accurate signaling of visual scenes. Understanding mechanisms behind luminance adaptation at different retinal levels is important for understanding how the retina functions in a dynamic environment. In the mouse, we show that dopamine pathways reduce inner retinal inhibition similar to increased background luminance, suggesting the two are linked and highlighting a possible mechanism for light adaptation at an early retinal processing center.

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.

Sensory systems must avoid saturation to encode a wide range of stimulus intensities. One way the retina accomplishes this is by using both dim-light-sensing rod and bright-light-sensing cone photoreceptor circuits. OFF cone bipolar cells are a key point in this process, as they receive both excitatory input from cones and inhibitory input from AII amacrine cells via the rod pathway. However, in addition to AII amacrine cell input, other inhibitory inputs from cone pathways also modulate OFF cone bipolar cell light signals. It is unknown how these inhibitory inputs to OFF cone bipolar cells change when switching between rod and cone pathways or whether all OFF cone bipolar cells receive rod pathway input. We found that one group of OFF cone bipolar cells (types 1, 2, and 4) receive rod-mediated inhibitory inputs that likely come from the rod-AII amacrine cell pathway, while another group of OFF cone bipolar cells (type 3) do not. In both cases, dark-adapted rod-dominant light responses showed a significant contribution of glycinergic inhibition, which decreased with light adaptation and was, surprisingly, compensated by an increase in GABAergic inhibition. As GABAergic input has distinct timing and spatial spread from glycinergic input, a shift from glycinergic to GABAergic inhibition could significantly alter OFF cone bipolar cell signaling to downstream OFF ganglion cells. Larger GABAergic input could reflect an adjustment of OFF bipolar cell spatial inhibition, which may be one mechanism that contributes to retinal spatial sensitivity in the light.

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 Ca2+ buffering or blocking prolonged L-type Ca2+ channels and Ca2+ release from intracellular stores. Our results suggest that GABAergic amacrine cells have a prolonged buildup of Ca2+ 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.

The retina adjusts its signaling gain over a wide range of light levels. A functional result of this is increased visual acuity at brighter luminance levels (light adaptation) due to shifts in the excitatory center-inhibitory surround receptive field parameters of ganglion cells that increases their sensitivity to smaller light stimuli. Recent work supports the idea that changes in ganglion cell spatial sensitivity with background luminance are due in part to inner retinal mechanisms, possibly including modulation of inhibition onto bipolar cells. To determine how the receptive fields of OFF cone bipolar cells may contribute to changes in ganglion cell resolution, the spatial extent and magnitude of inhibitory and excitatory inputs were measured from OFF bipolar cells under dark- and light-adapted conditions. There was no change in the OFF bipolar cell excitatory input with light adaptation; however, the spatial distributions of inhibitory inputs, including both glycinergic and GABAergic sources, became significantly narrower, smaller, and more transient. The magnitude and size of the OFF bipolar cell center-surround receptive fields as well as light-adapted changes in resting membrane potential were incorporated into a spatial model of OFF bipolar cell output to the downstream ganglion cells, which predicted an increase in signal output strength with light adaptation. We show a prominent role for inner retinal spatial signals in modulating the modeled strength of bipolar cell output to potentially play a role in ganglion cell visual sensitivity and acuity.

Dark- and light-adaptation of retinal neurons allows our vision to operate over an enormous light intensity range. Here we report a novel mechanism which controls the light sensitivity and operational range of rod-driven bipolar cells that mediate dim-light vision. Our data indicate that the light responses of these cells are enhanced by sustained chloride currents via GABAC receptor channels. This sensitizing GABAergic input is controlled by dopamine D1 receptors, with horizontal cells serving as a plausible source of GABA release. Our findings expand the role of dopamine in vision from its well-established function of suppressing rod-driven signals in bright light to enhancing the same signals under dim illumination. They further reveal a novel role for GABA in sensitizing the circuitry for dim-light vision, thereby complementing GABA’s traditional role in providing dynamic feedforward and feedback inhibition in the retina.

Bipolar cells (BCs) are critical relay neurons in the retina that are organized into parallel signaling pathways. The three main signaling pathways in the mammalian retina are the rod, ON cone, and OFF cone BCs. Rod BCs mediate incrementing dim light signals from rods, and ON cone and OFF cone BCs mediate incrementing and decrementing brighter light signals from cones, respectively. The outputs of BCs are shaped by inhibitory inputs from GABAergic and glycinergic amacrine cells in the inner plexiform layer, mediated by three distinct types of inhibitory receptors: GABAA, GABAC, and glycine receptors. The three main BC pathways receive distinct forms of inhibition from these three receptors that shape their light-evoked inhibitory signals. Rod BC inhibition is dominated by slow GABAC receptor inhibition, while OFF cone BCs are dominated by glycinergic inhibition. The inhibitory inputs to BCs are also shaped by serial inhibitory connections between GABAergic amacrine cells that limit the spatial profile of BC inhibition. We discuss our recent studies on how inhibitory inputs to BCs are shaped by receptor expression, receptor properties, and neurotransmitter release properties and how these affect the output of BCs.

Diverse retinal outputs are mediated by ganglion cells that receive excitatory input from distinct classes of bipolar cells (BCs). These classes of BCs separate visual signals into rod, ON and OFF cone pathways. Although BC signalling is a major determinant of the ganglion cell-mediated retinal output, it is not fully understood how light-evoked, presynaptic inhibition from amacrine cell inputs shapes BC outputs. To determine whether differences in presynaptic inhibition uniquely modulate BC synaptic output to specific ganglion cells, we assessed the inhibitory contributions of GABAA, GABAC and glycine receptors across the BC pathways. Here we show that different proportions of GABAA and GABAC receptor-mediated inhibition determined the kinetics of GABAergic presynaptic inhibition across different BC classes. Large, slow GABAC and small, fast GABAA receptor-mediated inputs to rod BCs prolonged light-evoked inhibitory postsynaptic currents (L-IPSCs), while smaller GABAC and larger GABAA receptor-mediated contributions produced briefer L-IPSCs in ON and OFF cone BCs. Glycinergic inhibition also varied across BC class. In the rod-dominant conditions studied here, slow glycinergic inputs dominated L-IPSCs in OFF cone BCs, attributable to inputs from the rod pathway via AII amacrine cells, while rod and ON cone BCs received little and no glycinergic input, respectively. As these large glycinergic inputs come from rod signalling pathways, in cone-dominant conditions L-IPSCs in OFF cone bipolar cells will probably be dominated by GABAA receptor-mediated input. Thus, unique presynaptic receptor combinations mediate distinct forms of inhibition to selectively modulate BC outputs, enhancing the distinctions among parallel retinal signals.

Rod bipolar cells relay visual signals evoked by dim illumination from the outer to the inner retina. GABAergic and glycinergic amacrine cells contact rod bipolar cell terminals, where they modulate transmitter release and contribute to the receptive field properties of third order neurones. However, it is not known how these distinct inhibitory inputs affect rod bipolar cell output and subsequent retinal processing. To determine whether GABAA, GABAC and glycine receptors made different contributions to light-evoked inhibition, we recorded light-evoked inhibitory postsynaptic currents (L-IPSCs) from rod bipolar cells mediated by each pharmacologically isolated receptor. All three receptors contributed to L-IPSCs, but their relative roles differed; GABAC receptors transferred significantly more charge than GABAA and glycine receptors. We determined how these distinct inhibitory inputs affected rod bipolar cell output by recording light-evoked excitatory postsynaptic currents (L-EPSCs) from postsynaptic AII and A17 amacrine cells. Consistent with their relative contributions to L-IPSCs, GABAC receptor activation most effectively reduced the L-EPSCs, while glycine and GABAA receptor activation reduced the L-EPSCs to a lesser extent. We also found that GABAergic L-IPSCs in rod bipolar cells were limited by GABAA receptor-mediated inhibition between amacrine cells. We show that GABAA, GABAC and glycine receptors mediate functionally distinct inhibition to rod bipolar cells, which differentially modulated light-evoked rod bipolar cell output. Our findings suggest that modulating the relative proportions of these inhibitory inputs could change the characteristics of rod bipolar cell output.

Previously, we showed that ethanol increases synaptic glycine currents, an effect that depends on ethanol concentration and developmental age of the preparation. Glycine receptor (GlyR) subunits undergo a shift from α2/β to α1/β from neonate to juvenile ages, with synaptic glycine currents from neonate hypoglossal motoneurons (HMs) being less sensitive to ethanol than those from juvenile HMs. Here we investigate whether these dose and developmental effects are also present in excised membrane patches containing GlyRs and if ethanol changes response kinetics. We excised outside-out patches from rat HM somata and applied glycine using either a picospritzer or piezo stack translator. Ethanol (100 mM) increased the response to glycine (200 μM) of patches from neonate and juvenile HMs. However, 30 mM ethanol increased the response from only juvenile HM patches. Using a lower concentration of glycine (30 μM) to observe single channel openings, we found that 100 mM ethanol increased the number of GlyRs that open in response to glycine and decreased first latency to channel opening. To investigate GlyR kinetic properties, we rapidly applied 1 mM glycine for 1 ms and found that glycine currents were increased by ethanol (100 mM) at both ages. For patches from juvenile HMs, ethanol consistently decreased response rise-time and increased response decay time. Using kinetic modeling, we determined that ethanol's potentiation of the glycine response arises from an increase in the glycine association ( kon) and a decrease in the dissociation ( koff) rate constants, resulting in increased glycine affinity of the GlyR.

Neurotransmitter release varies between neurons due to differences in presynaptic mechanisms such as Ca2+ 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 Ca2+ 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 Ca2+. 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.

The retina responds to a wide range of light stimuli by adaptation of retinal signaling to background light intensity and the use of two different photoreceptors: rods that sense dim light and cones that sense bright light. Rods signal to rod bipolar cells that receive significant inhibition from amacrine cells in the dark, especially from a rod bipolar cell-activated GABAergic amacrine cell. This inhibition modulates the output of rod bipolar cells onto downstream neurons. However, it was not clear how the inhibition of rod bipolar cells changes when rod signaling is limited by an adapting background light and cone signaling becomes dominant. We found that both light-evoked and spontaneous rod bipolar cell inhibition significantly decrease with light adaptation. This suggests a global decrease in the activity of amacrine cells that provide input to rod bipolar cells with light adaptation. However, inhibition to rod bipolar cells is also limited by GABAergic connections between amacrine cells, which decrease GABAergic input to rod bipolar cells. When we removed this serial inhibition, the light-evoked inhibition to rod bipolar cells remained after light adaptation. These results suggest that decreased inhibition to rod bipolar cells after light adaptation is due to decreased rod pathway activity as well as an active increase in inhibition between amacrine cells. Together these serve to limit rod bipolar cell inhibition after light adaptation, when the rod pathway is inactive and modulation of the signal is not required. This suggests an efficiency mechanism in the retina to limit unnecessary signaling.

The visual system is highly sensitive to dynamic features in the visual scene. However, it is not known how or where this enhanced sensitivity first occurs. We investigated this phenomenon by studying interactions between excitatory and inhibitory synapses in the second synaptic layer of the mouse retina. We found that these interactions showed activity-dependent changes that enhanced signaling of dynamic stimuli. Excitatory signaling from cone bipolar cells to ganglion cells exhibited strong synaptic depression, attributable to reduced glutamate release from bipolar cells. This depression was relieved by amacrine cell inhibitory feedback that activated presynaptic GABACreceptors. We found that the balance between excitation and feedback inhibition depended on stimulus frequency; at short interstimulus intervals, excitation was enhanced, attributable to reduced inhibitory feedback. This dynamic interplay may enrich visual processing by enhancing retinal responses to closely spaced temporal events, representing rapid changes in the visual environment.

While connections between inhibitory interneurons are common circuit elements, it has been difficult to define their signal processing roles because of the inability to activate these circuits using natural stimuli. We overcame this limitation by studying connections between inhibitory amacrine cells in the retina. These interneurons form spatially extensive inhibitory networks that shape signaling between bipolar cell relay neurons to ganglion cell output neurons. We investigated how amacrine cell networks modulate these retinal signals by selectively activating the networks with spatially defined light stimuli. The roles of amacrine cell networks were assessed by recording their inhibitory synaptic outputs in bipolar cells that suppress bipolar cell output to ganglion cells. When the amacrine cell network was activated by large light stimuli, the inhibitory connections between amacrine cells unexpectedly depressed bipolar cell inhibition. Bipolar cell inhibition elicited by smaller light stimuli or electrically activated feedback inhibition was not suppressed because these stimuli did not activate the connections between amacrine cells. Thus the activation of amacrine cell circuits with large light stimuli can shape the spatial sensitivity of the retina by limiting the spatial extent of bipolar cell inhibition. Because inner retinal inhibition contributes to ganglion cell surround inhibition, in part, by controlling input from bipolar cells, these connections may refine the spatial properties of the retinal output. This functional role of interneuron connections may be repeated throughout the CNS.

Synaptic integration is modulated by inhibition onto the dendrites of postsynaptic cells. However, presynaptic inhibition at axonal terminals also plays a critical role in the regulation of neurotransmission. In contrast to the development of inhibitory synapses onto dendrites, GABAergic/glycinergic synaptogenesis onto axon terminals has not been widely studied. Because retinal bipolar cells receive subclass-specific patterns of GABAergic and glycinergic presynaptic inhibition, they are a good model for studying the development of inhibition at axon terminals. Here, using whole cell recording methods and transgenic mice in which subclasses of retinal bipolar cells are labeled, we determined the temporal sequence and patterning of functional GABAergic and glycinergic input onto the major subclasses of bipolar cells. We found that the maturation of GABAergic and glycinergic synapses onto the axons of rod bipolar cells (RBCs), on-cone bipolar cells (on-CBCs) and off-cone bipolar cells (off-CBCs) were temporally distinct: spontaneous chloride-mediated currents are present in RBCs earlier in development compared with on- and off-CBC, and RBCs receive GABAergic and glycinergic input simultaneously, whereas in off-CBCs, glycinergic transmission emerges before GABAergic transmission. Because on-CBCs show little inhibitory activity, GABAergic and glycinergic events could not be pharmacologically distinguished for these bipolar cells. The balance of GABAergic and glycinergic input that is unique to RBCs and off-CBCs is established shortly after the onset of synapse formation and precedes visual experience. Our data suggest that presynaptic modulation of glutamate transmission from bipolar cells matures rapidly and is differentially coordinated for GABAergic and glycinergic synapses onto distinct bipolar cell subclasses.

Synaptic inhibition is determined by the properties of postsynaptic receptors, neurotransmitter release, and clearance, but little is known about how these factors shape sensation-evoked inhibition. The retina is an ideal system to investigate inhibition because it can be activated physiologically with light, and separate inhibitory pathways can be assayed by recording from rod bipolar cells that possess distinct glycine, GABAA, and GABACreceptors (R). We show that receptor properties differentially shape spontaneous IPSCs, whereas both transmitter release and receptor properties shape light-evoked (L) IPSCs. GABACR-mediated IPSCs decayed the slowest, whereas glycineR- and GABAAR-mediated IPSCs decayed more rapidly. Slow GABACRs determined the L-IPSC decay, whereas GABAARs and glycineRs, which mediated rapid onset responses, determined the start of the L-IPSC. Both fast and slow inhibitory inputs distinctly shaped the output of rod bipolar cells. The slow GABACRs truncated glutamate release, making the A17 amacrine cell L-EPSCs more transient, whereas the fast GABAAR and glycineRs reduced the initial phase of glutamate release, limiting the peak amplitude of the L-EPSC. Estimates of transmitter release time courses suggested that glycine release was more prolonged than GABA release. The time course of GABA release activating GABACRs was slower than that activating GABAARs, consistent with spillover activation of GABACRs. Thus, both postsynaptic receptor and transmitter release properties shape light-evoked inhibition in retina.

Rod bipolar cells relay visual signals evoked by dim illumination from the outer to the inner retina. GABAergic and glycinergic amacrine cells contact rod bipolar cell terminals, where they modulate transmitter release and contribute to the receptive field properties of third order neurones. However, it is not known how these distinct inhibitory inputs affect rod bipolar cell output and subsequent retinal processing. To determine whether GABA(A), GABA(C) and glycine receptors made different contributions to light-evoked inhibition, we recorded light-evoked inhibitory postsynaptic currents (L-IPSCs) from rod bipolar cells mediated by each pharmacologically isolated receptor. All three receptors contributed to L-IPSCs, but their relative roles differed; GABA(C) receptors transferred significantly more charge than GABA(A) and glycine receptors. We determined how these distinct inhibitory inputs affected rod bipolar cell output by recording light-evoked excitatory postsynaptic currents (L-EPSCs) from postsynaptic AII and A17 amacrine cells. Consistent with their relative contributions to L-IPSCs, GABA(C) receptor activation most effectively reduced the L-EPSCs, while glycine and GABA(A) receptor activation reduced the L-EPSCs to a lesser extent. We also found that GABAergic L-IPSCs in rod bipolar cells were limited by GABA(A) receptor-mediated inhibition between amacrine cells. We show that GABA(A), GABA(C) and glycine receptors mediate functionally distinct inhibition to rod bipolar cells, which differentially modulated light-evoked rod bipolar cell output. Our findings suggest that modulating the relative proportions of these inhibitory inputs could change the characteristics of rod bipolar cell output.

Ethanol potentiates glycinergic synaptic transmission to hypoglossal motoneurons (HMs). This effect on glycinergic transmission changes with postnatal development in that juvenile HMs (P9–13) are more sensitive to ethanol than neonate HMs (P1–3). We have now extended our previous study to investigate ethanol modulation of synaptic GABAA receptors (GABAARs), because both GABA and glycine mediate inhibitory synaptic transmission to brain stem motoneurons. We tested the effects of ethanol on GABAergic and glycinergic miniature inhibitory postsynaptic currents (mIPSCs) recorded from neonate and juvenile rat HMs in an in vitro slice preparation. Bath application of 30 mM ethanol had no significant effect on the GABAergic mIPSC amplitude or frequency recorded at either age. At 100 mM, ethanol significantly decreased the GABAergic mIPSC amplitude recorded from neonate (6 ± 3%, P < 0.05) and juvenile (16 ± 3%, P < 0.01) HMs. The same concentration of ethanol increased the GABAergic mIPSC frequency recorded from neonate (64 ± 17%, P < 0.05) and juvenile (40 ± 15%, n.s.) HMs. In contrast, 100 mM ethanol robustly potentiated glycinergic mIPSC amplitude in neonate (31 ± 3%, P < 0.0001) and juvenile (41 ± 7%, P < 0.001) HMs. These results suggest that glycine receptors are more sensitive to modulation by ethanol than GABAA receptors and that 100 mM ethanol has the opposite effect on GABAAR-mediated currents in juvenile HMs, that is, inhibition rather than enhancement. Further, comparing ethanol's effects on GABAergic mIPSC amplitude and frequency, ethanol modulates GABAergic synaptic transmission to HMs differentially. Presynaptically, ethanol enhances mIPSC frequency while postsynaptically it decreases mIPSC amplitude.

During postnatal motoneuron development, the glycine receptor (GlyR) α subunit changes from α2 (fetal) to α1 (adult). To study the effect this change has on ethanol potentiation of GlyR currents in hypoglossal motoneurons (HMs), we placed neurons into two groups: neonate [ postnatal day 1 to 3( P1–3)], primarily expressing α2, and juvenile ( P9–13), primarily expressing α1. We found that glycinergic spontaneous miniature inhibitory postsynaptic currents (mIPSCs) in neonate HMs are less sensitive to ethanol than in juveniles. Thirty millimolar ethanol increased the amplitude of juvenile mIPSCs but did not significantly change neonatal mIPSCs. However, 100 mM ethanol increased the amplitudes of both neonate and juvenile mIPSCs. There was a significant difference between age groups in the average ethanol-induced increase in mIPSC amplitude for 10, 30, 50, and 100 mM ethanol. In both age groups ethanol increased the frequency of glycinergic mIPSCs, but there was no difference in the amount of frequency increase between age groups. Ethanol (100 mM) also potentiated evoked IPSCs (eIPSCs) in both neonate and juvenile HMs. As we observed for mIPSCs, 30 mM ethanol increased the amplitude of juvenile eIPSCs, but had no significant effect on eIPSCs in neonate HMs. Ethanol also potentiated currents induced by exogenously applied glycine in both neonate and juvenile HMs. These results suggest that ethanol directly modulates the GlyR. To investigate possible mechanisms for this, we analyzed the time course of mIPSCs and single-channel conductance of the GlyR in the presence and absence of ethanol. We found that ethanol did not significantly change the time course of mIPSCs. We also determined that ethanol did not significantly change the single-channel conductance of synaptic GlyRs, as estimated by nonstationary noise analysis of mIPSCs. We conclude that the adult form of the native GlyR is more sensitive to ethanol than the fetal form. Further, enhancement of GlyR currents involves mechanisms other than an increase in the single-channel conductance or factors that alter the decay kinetics.