Erika D. Eggers

GABAA, GABAC and glycine receptor‐mediated inhibition differentially affects light‐evoked signalling from mouse retinal rod bipolar cells

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.

GABAC receptor-mediated inhibition in the retina

Inhibition at bipolar cell axon terminals regulates excitatory signaling to ganglion cells and is mediated, in part, by GABAC receptors. We investigated GABAC receptor-mediated inhibition using pharmacological approaches and genetically altered mice that lack GABAC receptors. Responses to applied GABA showed distinct time courses in various bipolar cell classes, attributable to different proportions of GABAA and GABAC receptors. The elimination of GABAC receptors in GABAC null mice reduced and shortened GABA-activated currents and light-evoked inhibitory synaptic currents (L-IPSCs) in rod bipolar cells. ERG measurements and recordings from the optic nerve showed that inner retinal function was altered in GABAC null mice. These data suggest that GABAC receptors determine the time course and extent of inhibition at bipolar cell terminals that, in turn, modulates the magnitude of excitatory transmission from bipolar cells to ganglion cells.

Presynaptic inhibition differentially shapes transmission in distinct circuits in the mouse retina

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 vision is controlled by dopamine-dependent sensitization of rod bipolar cells by GABA

Dark and light adaptation of retinal neurons allow our vision to operate over an enormous light intensity range. Here we report a mechanism that 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 GABA(C) 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 role for GABA in sensitizing the circuitry for dim-light vision, thereby complementing GABAs traditional role in providing dynamic feedforward and feedback inhibition in the retina.

Receptor and Transmitter Release Properties Set the Time Course of Retinal Inhibition

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 GABAC receptors (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.

Multiple pathways of inhibition shape bipolar cell responses 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: GABA(A), GABA(C), 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 GABA(C) 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.

Interneuron Circuits Tune Inhibition in Retinal Bipolar Cells

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.

Development of presynaptic inhibition onto retinal bipolar cell axon terminals is subclass-specific.

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.

Nonlinear Interactions between Excitatory and Inhibitory Retinal Synapses Control Visual Output

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 GABAC receptors. 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.

Inhibition to retinal rod bipolar cells is regulated by light levels

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.