Restoration of vision after de novo genesis of rod photoreceptors in mammalian retinas.

In zebrafish, Müller glia (MG) are a source of retinal stem cells that can replenish damaged retinal neurons and restore vision1. In mammals, however, MG do not spontaneously re-enter the cell cycle to generate a population of stem or progenitor cells that differentiate into retinal neurons. Nevertheless, the regenerative machinery may exist in the mammalian retina, as retinal injury can stimulate MG proliferation followed by limited neurogenesis2-7. Therefore, there is still a fundamental question regarding whether MG-derived regeneration can be exploited to restore vision in mammalian retinas. Gene transfer of ß-catenin stimulates MG proliferation in the absence of injury in mouse retinas8. Here we report that following gene transfer of ß-catenin, cell-cycle-reactivated MG can be reprogrammed to generate rod photoreceptors by subsequent gene transfer of transcription factors essential for rod cell fate specification and determination. MG-derived rods restored visual responses in Gnat1rd17Gnat2cpfl3 double mutant mice, a model of congenital blindness9,10, throughout the visual pathway from the retina to the primary visual cortex. Together, our results provide evidence of vision restoration after de novo MG-derived genesis of rod photoreceptors in mammalian retinas.

GABA release selectively regulates synapse development at distinct inputs on direction-selective retinal ganglion cells.

Synaptic inhibition controls a neuron's output via functionally distinct inputs at two subcellular compartments, the cell body and the dendrites. It is unclear whether the assembly of these distinct inhibitory inputs can be regulated independently by neurotransmission. In the mammalian retina, γ-aminobutyric acid (GABA) release from starburst amacrine cells (SACs) onto the dendrites of on-off direction-selective ganglion cells (ooDSGCs) is essential for directionally selective responses. We found that ooDSGCs also receive GABAergic input on their somata from other amacrine cells (ACs), including ACs containing the vasoactive intestinal peptide (VIP). When net GABAergic transmission is reduced, somatic, but not dendritic, GABAA receptor clusters on the ooDSGC increased in number and size. Correlative fluorescence imaging and serial electron microscopy revealed that these enlarged somatic receptor clusters are localized to synapses. By contrast, selectively blocking vesicular GABA release from either SACs or VIP ACs did not alter dendritic or somatic receptor distributions on the ooDSGCs, showing that neither SAC nor VIP AC GABA release alone is required for the development of inhibitory synapses in ooDSGCs. Furthermore, a reduction in net GABAergic transmission, but not a selective reduction from SACs, increased excitatory drive onto ooDSGCs. This increased excitation may drive a homeostatic increase in ooDSGC somatic GABAA receptors. Differential regulation of GABAA receptors on the ooDSGC's soma and dendrites could facilitate homeostatic control of the ooDSGC's output while enabling the assembly of the GABAergic connectivity underlying direction selectivity to be indifferent to altered transmission.

Convergence and Divergence of CRH Amacrine Cells in Mouse Retinal Circuitry.

Inhibitory interneurons sculpt the outputs of excitatory circuits to expand the dynamic range of information processing. In mammalian retina, >30 types of amacrine cells provide lateral inhibition to vertical, excitatory bipolar cell circuits, but functional roles for only a few amacrine cells are well established. Here, we elucidate the function of corticotropin-releasing hormone (CRH)-expressing amacrine cells labeled in Cre-transgenic mice of either sex. CRH cells costratify with the ON alpha ganglion cell, a neuron highly sensitive to positive contrast. Electrophysiological and optogenetic analyses demonstrate that two CRH types (CRH-1 and CRH-3) make GABAergic synapses with ON alpha cells. CRH-1 cells signal via graded membrane potential changes, whereas CRH-3 cells fire action potentials. Both types show sustained ON-type responses to positive contrast over a range of stimulus conditions. Optogenetic control of transmission at CRH-1 synapses demonstrates that these synapses are tuned to low temporal frequencies, maintaining GABA release during fast hyperpolarizations during brief periods of negative contrast. CRH amacrine cell output is suppressed by prolonged negative contrast, when ON alpha ganglion cells continue to receive inhibitory input from converging OFF-pathway amacrine cells; the converging ON- and OFF-pathway inhibition balances tonic excitatory drive to ON alpha cells. Previously, it was demonstrated that CRH-1 cells inhibit firing by suppressed-by-contrast (SbC) ganglion cells during positive contrast. Therefore, divergent outputs of CRH-1 cells inhibit two ganglion cell types with opposite responses to positive contrast. The opposing responses of ON alpha and SbC ganglion cells are explained by differing excitation/inhibition balance in the two circuits.SIGNIFICANCE STATEMENT A goal of neuroscience research is to explain the function of neural circuits at the level of specific cell types. Here, we studied the function of specific types of inhibitory interneurons, corticotropin-releasing hormone (CRH) amacrine cells, in the mouse retina. Genetic tools were used to identify and manipulate CRH cells, which make GABAergic synapses with a well studied ganglion cell type, the ON alpha cell. CRH cells converge with other types of amacrine cells to tonically inhibit ON alpha cells and balance their high level of excitation. CRH cells diverge to different types of ganglion cell, the unique properties of which depend on their balance of excitation and inhibition.

Function and Circuitry of VIP+ Interneurons in the Mouse Retina.

Visual processing in the retina depends on coordinated signaling by interneurons. Photoreceptor signals are relayed to ∼20 ganglion cell types through a dozen excitatory bipolar interneurons, each responsive to light increments (ON) or decrements (OFF). ON and OFF bipolar cell pathways become tuned through specific connections with inhibitory interneurons: horizontal and amacrine cells. A major obstacle for understanding retinal circuitry is the unknown function of most of the ∼30-40 amacrine cell types, each of which synapses onto a subset of bipolar cell terminals, ganglion cell dendrites, and other amacrine cells. Here, we used a transgenic mouse line in which vasoactive intestinal polypeptide-expressing (VIP+) GABAergic interneurons express Cre recombinase. Targeted whole-cell recordings of fluorescently labeled VIP+ cells revealed three predominant types: wide-field bistratified and narrow-field monostratified cells with somas in the inner nuclear layer (INL) and medium-field monostratified cells with somas in the ganglion cell layer (GCL). Bistratified INL cells integrated excitation and inhibition driven by both ON and OFF pathways with little spatial tuning. Narrow-field INL cells integrated excitation driven by the ON pathway and inhibition driven by both pathways, with pronounced hyperpolarizations at light offset. Monostratified GCL cells integrated excitation and inhibition driven by the ON pathway and showed center-surround spatial tuning. Optogenetic experiments showed that, collectively, VIP+ cells made strong connections with OFF δ, ON-OFF direction-selective, and W3 ganglion cells but weak, inconsistent connections with ON and OFF α cells. Revealing VIP+ cell morphologies, receptive fields and synaptic connections advances our understanding of their role in visual processing. SIGNIFICANCE STATEMENT: The retina is a model system for understanding nervous system function. At the first stage, rod and cone photoreceptors encode light and communicate with a complex network of interneurons. These interneurons drive the responses of ganglion cells, which form the optic nerve and transmit visual information to the brain. Presently, we lack information about many of the retina's inhibitory amacrine interneurons. In this study, we used genetically modified mice to study the light responses and intercellular connections of specific amacrine cell types. The results show diversity in the shape and function of the studied amacrine cells and elucidate their connections with specific types of ganglion cell. The findings advance our understanding of the cellular basis for retinal function.

Developmental changes in NMDA receptor subunit composition at ON and OFF bipolar cell synapses onto direction-selective retinal ganglion cells.

In the developing mouse retina, spontaneous and light-driven activity shapes bipolar→ganglion cell glutamatergic synapse formation, beginning around the time of eye-opening (P12-P14) and extending through the first postnatal month. During this time, glutamate release can spill outside the synaptic cleft and possibly stimulate extrasynaptic NMDA-type glutamate receptors (NMDARs) on ganglion cells. Furthermore, the role of NMDARs during development may differ between ON and OFF bipolar synapses as in mature retina, where ON synapses reportedly include extrasynaptic NMDARs with GluN2B subunits. To better understand the function of glutamatergic synapses during development, we made whole-cell recordings of NMDAR-mediated responses, in vitro, from two types of genetically identified direction-selective ganglion cells (dsGCs): TRHR (thyrotropin-releasing hormone receptor) and Drd4 (dopamine receptor 4). Both dsGC types responded to puffed NMDA between P7 and P28; and both types exhibited robust light-evoked NMDAR-mediated responses at P14 and P28 that were quantified by conductance analysis during nicotinic and GABA(A) receptor blockade. For a given cell type and at a given age, ON and OFF bipolar cell inputs evoked similar NMDAR-mediated responses, suggesting that ON-versus-OFF differences in mature retina do not apply to the cell types or ages studied here. At P14, puff- and light-evoked NMDAR-mediated responses in both dsGCs were partially blocked by the GluN2B antagonist ifenprodil, whereas at P28 only TRHR cells remained ifenprodil-sensitive. NMDARs contribute at both ON and OFF bipolar cell synapses during a period of robust activity-dependent synaptic development, with declining GluN2B involvement over time in specific ganglion cell types.

Excitatory synaptic inputs to mouse on-off direction-selective retinal ganglion cells lack direction tuning.

Direction selectivity represents a fundamental visual computation. In mammalian retina, On-Off direction-selective ganglion cells (DSGCs) respond strongly to motion in a preferred direction and weakly to motion in the opposite, null direction. Electrical recordings suggested three direction-selective (DS) synaptic mechanisms: DS GABA release during null-direction motion from starburst amacrine cells (SACs) and DS acetylcholine and glutamate release during preferred direction motion from SACs and bipolar cells. However, evidence for DS acetylcholine and glutamate release has been inconsistent and at least one bipolar cell type that contacts another DSGC (On-type) lacks DS release. Here, whole-cell recordings in mouse retina showed that cholinergic input to On-Off DSGCs lacked DS, whereas the remaining (glutamatergic) input showed apparent DS. Fluorescence measurements with the glutamate biosensor intensity-based glutamate-sensing fluorescent reporter (iGluSnFR) conditionally expressed in On-Off DSGCs showed that glutamate release in both On- and Off-layer dendrites lacked DS, whereas simultaneously recorded excitatory currents showed apparent DS. With GABA-A receptors blocked, both iGluSnFR signals and excitatory currents lacked DS. Our measurements rule out DS release from bipolar cells onto On-Off DSGCs and support a theoretical model suggesting that apparent DS excitation in voltage-clamp recordings results from inadequate voltage control of DSGC dendrites during null-direction inhibition. SAC GABA release is the apparent sole source of DS input onto On-Off DSGCs.

Receptive field properties of bipolar cell axon terminals in direction-selective sublaminas of the mouse retina.

Retinal bipolar cells (BCs) transmit visual signals in parallel channels from the outer to the inner retina, where they provide glutamatergic inputs to specific networks of amacrine and ganglion cells. Intricate network computation at BC axon terminals has been proposed as a mechanism for complex network computation, such as direction selectivity, but direct knowledge of the receptive field property and the synaptic connectivity of the axon terminals of various BC types is required in order to understand the role of axonal computation by BCs. The present study tested the essential assumptions of the presynaptic model of direction selectivity at axon terminals of three functionally distinct BC types that ramify in the direction-selective strata of the mouse retina. Results from two-photon Ca(2+) imaging, optogenetic stimulation, and dual patch-clamp recording demonstrated that 1) CB5 cells do not receive fast GABAergic synaptic feedback from starburst amacrine cells (SACs); 2) light-evoked and spontaneous Ca(2+) responses are well coordinated among various local regions of CB5 axon terminals; 3) CB5 axon terminals are not directionally selective; 4) CB5 cells consist of two novel functional subtypes with distinct receptive field structures; 5) CB7 cells provide direct excitatory synaptic inputs to, but receive no direct GABAergic synaptic feedback from, SACs; and 6) CB7 axon terminals are not directionally selective, either. These findings help to simplify models of direction selectivity by ruling out complex computation at BC terminals. They also show that CB5 comprises two functional subclasses of BCs.

Divisive suppression explains high-precision firing and contrast adaptation in retinal ganglion cells.

Visual processing depends on specific computations implemented by complex neural circuits. Here, we present a circuit-inspired model of retinal ganglion cell computation, targeted to explain their temporal dynamics and adaptation to contrast. To localize the sources of such processing, we used recordings at the levels of synaptic input and spiking output in the in vitro mouse retina. We found that an ON-Alpha ganglion cell's excitatory synaptic inputs were described by a divisive interaction between excitation and delayed suppression, which explained nonlinear processing that was already present in ganglion cell inputs. Ganglion cell output was further shaped by spike generation mechanisms. The full model accurately predicted spike responses with unprecedented millisecond precision, and accurately described contrast adaptation of the spike train. These results demonstrate how circuit and cell-intrinsic mechanisms interact for ganglion cell function and, more generally, illustrate the power of circuit-inspired modeling of sensory processing.

Complexin 3 Increases the Fidelity of Signaling in a Retinal Circuit by Regulating Exocytosis at Ribbon Synapses.

Complexin (Cplx) proteins modulate the core SNARE complex to regulate exocytosis. To understand the contributions of Cplx to signaling in a well-characterized neural circuit, we investigated how Cplx3, a retina-specific paralog, shapes transmission at rod bipolar (RB)→AII amacrine cell synapses in the mouse retina. Knockout of Cplx3 strongly attenuated fast, phasic Ca(2+)-dependent transmission, dependent on local [Ca(2+)] nanodomains, but enhanced slower Ca(2+)-dependent transmission, dependent on global intraterminal [Ca(2+)] ([Ca(2+)]I). Surprisingly, coordinated multivesicular release persisted at Cplx3(-/-) synapses, although its onset was slowed. Light-dependent signaling at Cplx3(-/-) RB→AII synapses was sluggish, owing largely to increased asynchronous release at light offset. Consequently, propagation of RB output to retinal ganglion cells was suppressed dramatically. Our study links Cplx3 expression with synapse and circuit function in a specific retinal pathway and reveals a role for asynchronous release in circuit gain control.