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.

Ribbon Synapses and Retinal Disease: Review.

Synaptic ribbons are presynaptic protein complexes that are believed to be important for the transmission of sensory information in the visual system. Ribbons are selectively associated with those synapses where graded changes in membrane potential drive continuous neurotransmitter release. Defective synaptic transmission can arise as a result of the mutagenesis of a single ribbon component. Visual diseases that stem from malfunctions in the presynaptic molecular machinery of ribbon synapses in the retina are rare. In this review, we provide an overview of synaptopathies that give rise to retinal malfunction and our present understanding of the mechanisms that underlie their pathogenesis and discuss muscular dystrophies that exhibit ribbon synapse involvement in the pathology.

Correction: Mesnard et al. Eliminating Synaptic Ribbons from Rods and Cones Halves the Releasable Vesicle Pool and Slows Down Replenishment. Int. J. Mol. Sci. 2022, 23, 6429

The authors wish to make the following corrections to this paper [...].

Eliminating Synaptic Ribbons from Rods and Cones Halves the Releasable Vesicle Pool and Slows Down Replenishment.

Glutamate release from rod and cone photoreceptor cells involves presynaptic ribbons composed largely of the protein RIBEYE. To examine roles of ribbons in rods and cones, we studied mice in which GCamP3 replaced the B-domain of RIBEYE. We discovered that ribbons were absent from rods and cones of both knock-in mice possessing GCamP3 and conditional RIBEYE knockout mice. The mice lacking ribbons showed reduced temporal resolution and contrast sensitivity assessed with optomotor reflexes. ERG recordings showed 50% reduction in scotopic and photopic b-waves. The readily releasable pool (RRP) of vesicles in rods and cones measured using glutamate transporter anion currents (IA(glu)) was also halved. We also studied the release from cones by stimulating them optogenetically with ChannelRhodopsin2 (ChR2) while recording postsynaptic currents in horizontal cells. Recovery of the release from paired pulse depression was twofold slower in the rods and cones lacking ribbons. The release from rods at -40 mV in darkness involves regularly spaced multivesicular fusion events. While the regular pattern of release remained in the rods lacking ribbons, the number of vesicles comprising each multivesicular event was halved. Our results support conclusions that synaptic ribbons in rods and cones expand the RRP, speed up vesicle replenishment, and augment some forms of multivesicular release. Slower replenishment and a smaller RRP in photoreceptors lacking ribbons may contribute to diminished temporal frequency responses and weaker contrast sensitivity.

Direct Observation of Vesicle Transport on the Synaptic Ribbon Provides Evidence That Vesicles Are Mobilized and Prepared Rapidly for Release.

Synaptic ribbons are thought to provide vesicles for continuous release in some retinal nonspiking neurons, yet recent studies indicate that genetic removal of the ribbon has little effect on release kinetics. To investigate vesicle replenishment at synaptic ribbons, we used total internal reflection fluorescence microscopy to image synaptic vesicles and ribbons in retinal bipolar cells of goldfish (Carassius auratus) of both sexes. Analysis of vesicles released by trains of 30 ms depolarizations revealed that most releasable vesicles reside within 300 nm of the ribbon center. A single 30 ms step to 0 mV was sufficient to deplete the membrane-proximal vesicle pool, while triggering rapid stepwise movements of distal vesicles along the ribbon and toward the plasma membrane. Replenishment only becomes rate-limiting for recovery from paired-pulse depression for interstimulus intervals shorter than 250 ms. For longer interstimulus intervals, vesicle movement down the ribbon is fast enough to replenish released vesicles, but newly arrived vesicles are not release-ready. Notably, the rates of vesicle resupply and maturation of newcomers are among the fastest measured optically at any synapse. Lastly, our data show that the delay in vesicle departure increases and vesicle speed decreases with multiple stimuli. Our results support a role for ribbons in the supply of vesicles for release, provide direct measurements of vesicle movement down the ribbon, and suggest that multiple factors contribute to paired-pulse depression.SIGNIFICANCE STATEMENT Synaptic ribbons are macromolecular scaffolds that tether synaptic vesicles close to release sites in nonspiking neurons of the retina and cochlea. Because these neurons release neurotransmitter continuously, synaptic ribbons are assumed to act as platforms for supplying vesicles rapidly in the face of prolonged stimulation. Yet, ribbon synapses suffer from profound paired-pulse depression, which takes seconds to subside. We investigated the mechanistic origin of this phenomenon by directly imaging triggered vesicle movement and release at ribbon sites in retinal bipolar cells, and find that, although ribbon synapses deliver and prime vesicles faster than most conventional synapses, both vesicle absence and vesicle priming contribute to the long recovery from paired-pulse depression.

Function and regulation of TRPP2 ion channel revealed by a gain-of-function mutant.

Mutations in polycystin-1 and transient receptor potential polycystin 2 (TRPP2) account for almost all clinically identified cases of autosomal dominant polycystic kidney disease (ADPKD), one of the most common human genetic diseases. TRPP2 functions as a cation channel in its homomeric complex and in the TRPP2/polycystin-1 receptor/ion channel complex. The activation mechanism of TRPP2 is unknown, which significantly limits the study of its function and regulation. Here, we generated a constitutively active gain-of-function (GOF) mutant of TRPP2 by applying a mutagenesis scan on the S4-S5 linker and the S5 transmembrane domain, and studied functional properties of the GOF TRPP2 channel. We found that extracellular divalent ions, including Ca(2+), inhibit the permeation of monovalent ions by directly blocking the TRPP2 channel pore. We also found that D643, a negatively charged amino acid in the pore, is crucial for channel permeability. By introducing single-point ADPKD pathogenic mutations into the GOF TRPP2, we showed that different mutations could have completely different effects on channel activity. The in vivo function of the GOF TRPP2 was investigated in zebrafish embryos. The results indicate that, compared with wild type (WT), GOF TRPP2 more efficiently rescued morphological abnormalities, including curly tail and cyst formation in the pronephric kidney, caused by down-regulation of endogenous TRPP2 expression. Thus, we established a GOF TRPP2 channel that can serve as a powerful tool for studying the function and regulation of TRPP2. The GOF channel may also have potential application for developing new therapeutic strategies for ADPKD.

Global Ca2+ signaling drives ribbon-independent synaptic transmission at rod bipolar cell synapses.

Ribbon-type presynaptic active zones are a hallmark of excitatory retinal synapses, and the ribbon organelle is thought to serve as the organizing point of the presynaptic active zone. Imaging of exocytosis from isolated retinal neurons, however, has revealed ectopic release (i.e., release away from ribbons) in significant quantities. Here, we demonstrate in an in vitro mouse retinal slice preparation that ribbon-independent release from rod bipolar cells activates postsynaptic AMPARs on AII amacrine cells. This form of release appears to draw on a unique, ribbon-independent, vesicle pool. Experimental, anatomical, and computational analyses indicate that it is elicited by a significant, global elevation of intraterminal [Ca(2+)] arising following local buffer saturation. Our observations support the conclusion that ribbon-independent release provides a read-out of the average behavior of all of the active zones in a rod bipolar cell's terminal.

Amperometric resolution of a prespike stammer and evoked phases of fast release from retinal bipolar cells.

The neurotransmitter glutamate is used by most neurons in the brain to activate a multitude of different types of glutamate receptors and transporters involved in fast and relatively slower signaling. Synaptic ribbons are large presynaptic structures found in neurons involved in vision, balance, and hearing, which use a large number of glutamate-filled synaptic vesicles to meet their signaling demands. To directly measure synaptic vesicle release events, the ribbon-type presynaptic terminals of goldfish retinal bipolar cells were coaxed to release a false transmitter that could be monitored with amperometry by placing the carbon fiber directly on the larger synaptic terminal. Spontaneous secretion events formed a unimodal charge distribution, but single spike properties were heterogeneous. Larger events rose exponentially without interruption (τ ∼ 30 µs), and smaller events exhibited a stammer in their rising phase that is interpreted as a brief pause in pore dilation, a characteristic commonly associated with large dense core granule fusion pores. These events were entirely Ca(2+)-dependent. Holding the cells at -60 mV halted spontaneous release; and when the voltage was stepped to >-40 mV, secretion ensued. When stepping the voltage to 0 mV, novel kinetic phases of vesicle recruitment were revealed. Approximately 14 vesicles were released per ribbon in two kinetic phases with time constants of 1.5 and 16 ms, which are proposed to represent different primed states within the population of docked vesicles.

Properties of ribbon and non-ribbon release from rod photoreceptors revealed by visualizing individual synaptic vesicles.

Vesicle release from rod photoreceptors is regulated by Ca(2+) entry through L-type channels located near synaptic ribbons. We characterized sites and kinetics of vesicle release in salamander rods by using total internal reflection fluorescence microscopy to visualize fusion of individual synaptic vesicles. A small number of vesicles were loaded by brief incubation with FM1-43 or a dextran-conjugated, pH-sensitive form of rhodamine, pHrodo. Labeled organelles matched the diffraction-limited size of fluorescent microspheres and disappeared rapidly during stimulation. Consistent with fusion, depolarization-evoked vesicle disappearance paralleled electrophysiological release kinetics and was blocked by inhibiting Ca(2+) influx. Rods maintained tonic release at resting membrane potentials near those in darkness, causing depletion of membrane-associated vesicles unless Ca(2+) entry was inhibited. This depletion of release sites implies that sustained release may be rate limited by vesicle delivery. During depolarizing stimulation, newly appearing vesicles approached the membrane at ∼800 nm/s, where they paused for ∼60 ms before fusion. With fusion, vesicles advanced ∼18 nm closer to the membrane. Release events were concentrated near ribbons, but lengthy depolarization also triggered release from more distant non-ribbon sites. Consistent with greater contributions from non-ribbon sites during lengthier depolarization, damaging the ribbon by fluorophore-assisted laser inactivation (FALI) of Ribeye caused only weak inhibition of exocytotic capacitance increases evoked by 200-ms depolarizing test steps, whereas FALI more strongly inhibited capacitance increases evoked by 25 ms steps. Amplifying release by use of non-ribbon sites when rods are depolarized in darkness may improve detection of decrements in release when they hyperpolarize to light.

Patch-clamp recordings from lateral line neuromast hair cells of the living zebrafish.

Zebrafish are popular models for biological discovery. For investigators of the auditory and vestibular periphery, manipulations of hair cell and synaptic mechanisms have relied on inferences from extracellular recordings of physiological activity. We now provide data showing that hair cells and supporting cells of the lateral line can be directly patch-clamped, providing the first recordings of ionic channel activity, synaptic vesicle release, and gap junctional coupling in the neuromasts of living fish. Such capabilities will allow more detailed understanding of mechano-sensation of the zebrafish.

Presynaptic Proteins and Their Roles in Visual Processing by the Retina.

The sense of vision begins in the retina, where light is detected and processed through a complex series of synaptic connections into meaningful information relayed to the brain via retinal ganglion cells. Light responses begin as tonic and graded signals in photoreceptors, later emerging from the retina as a series of spikes from ganglion cells. Processing by the retina extracts critical features of the visual world, including spatial frequency, temporal frequency, motion direction, color, contrast, and luminance. To achieve this, the retina has evolved specialized and unique synapse types. These include the ribbon synapses of photoreceptors and bipolar cells, the dendritic synapses of amacrine and horizontal cells, and unconventional synaptic feedback from horizontal cells to photoreceptors. We review these unique synapses in the retina with a focus on the presynaptic molecules and physiological properties that shape their capabilities.

Role of Ribeye PXDLS/T-binding cleft in normal synaptic ribbon function.

Non-spiking sensory hair cells of the auditory and vestibular systems encode a dynamic range of graded signals with high fidelity by vesicle exocytosis at ribbon synapses. Ribeye, the most abundant protein in the synaptic ribbon, is composed of a unique A domain specific for ribbons and a B-domain nearly identical to the transcriptional corepressor CtBP2. CTBP2 and the B-domain of Ribeye contain a surface cleft that binds to proteins harboring a PXDLS/T peptide motif. Little is known about the importance of this binding site in synaptic function. Piccolo has a well-conserved PVDLT motif and we find that overexpressed Ribeye exhibits striking co-localization with Piccolo in INS-cells, while two separate mutants containing mutations in PXDLS/T-binding region, fail to co-localize with Piccolo. Similarly, co-transfected Ribeye and a piccolo fragment containing the PVDLT region co-localize in HEK cells. Expression of wild-type Ribeye-YFP in zebrafish neuromast hair cells returns electron densities to ribbon structures and mostly rescued normal synaptic transmission and morphological phenotypes in a mutant zebrafish lacking most Ribeye. By contrast, Ribeye-YFP harboring a mutation in the PXDLS/T-binding cleft resulted in ectopic electron dense aggregates that did not collect vesicles and the persistence of ribbons lacking electron densities. Furthermore, overexpression failed to return capacitance responses to normal levels. These results point toward a role for the PXDLS/T-binding cleft in the recruitment of Ribeye to ribbons and in normal synaptic function.

The Effects of Aging on Rod Bipolar Cell Ribbon Synapses.

The global health concern posed by age-related visual impairment highlights the need for further research focused on the visual changes that occur during the process of aging. To date, multiple sensory alterations related to aging have been identified, including morphological and functional changes in inner hair cochlear cells, photoreceptors, and retinal ganglion cells. While some age-related morphological changes are known to occur in rod bipolar cells in the retina, their effects on these cells and on their connection to other cells via ribbon synapses remain elusive. To investigate the effects of aging on rod bipolar cells and their ribbon synapses, we compared synaptic calcium currents, calcium dynamics, and exocytosis in zebrafish (Danio rerio) that were middle-aged (MA,18 months) or old-aged (OA, 36 months). The bipolar cell terminal in OA zebrafish exhibited a two-fold reduction in number of synaptic ribbons, an increased ribbon length, and a decrease in local Ca2+ signals at the tested ribbon location, with little change in the overall magnitude of the calcium current or exocytosis in response to brief pulses. Staining of the synaptic ribbons with antibodies specific for PKCa revealed shortening of the inner nuclear and plexiform layers (INL and IPL). These findings shed light on age-related changes in the retina that are related to synaptic ribbons and calcium signals.

High-resolution optical imaging of zebrafish larval ribbon synapse protein RIBEYE, RIM2, and CaV 1.4 by stimulation emission depletion microscopy.

The synaptic ribbon is a unique presynaptic structure with an intricate morphology in photoreceptors. Because of the resolution limit in conventional fluorescence microscopy, investigating ribbon protein locations has been challenging, especially in the early development stages of model animals. Here, we used stimulated emission depletion microscopy, a super-resolution imaging technique, to look at retina sections in 4 days post-fertilization (dpf) zebrafish. We observed that in photoreceptor cells, RIBEYE and RIM2 are expressed along the synaptic ribbon, with RIM2 consistently located inside of the horseshoe-shaped synaptic ribbon structure with RIBEYE located on the outside. The L-type calcium channel subunit, CACNA1F, exhibited small spot-like staining beneath the RIM2 and RIBEYE structures. Using morpholino antisense oligonucleotides to knock down RIBEYE expression, we observed fewer and shorter ribbons in the photoreceptor outer plexiform layers of 4 dpf fish retina as well as a reduction in RIM2 expression. The clustering of CACNA1F in these blind fish was no longer observed, but instead showed a diffuse expression in the photoreceptor terminal.

Rapid propagation of membrane tension at retinal bipolar neuron presynaptic terminals.

Many cellular activities, such as cell migration, cell division, phagocytosis, and exo-endocytosis, generate and are regulated by membrane tension gradients. Membrane tension gradients drive membrane flows, but there is controversy over how rapidly plasma membrane flow can relax tension gradients. Here, we show that membrane tension can propagate rapidly or slowly, spanning orders of magnitude in speed, depending on the cell type. In a neuronal terminal specialized for rapid synaptic vesicle turnover, membrane tension equilibrates within seconds. By contrast, membrane tension does not propagate in neuroendocrine adrenal chromaffin cells secreting catecholamines. Stimulation of exocytosis causes a rapid, global decrease in the synaptic terminal membrane tension, which recovers slowly due to endocytosis. Thus, membrane flow and tension equilibration may be adapted to distinct membrane recycling requirements.

Multimodal imaging of synaptic vesicles with a single probe.

A complete understanding of synaptic-vesicle recycling requires the use of multiple microscopy methods to obtain complementary information. However, many currently available probes are limited to a specific microscopy modality, which necessitates the use of multiple probes and labeling paradigms. Given the complexity of vesicle populations and recycling pathways, having new single-vesicle probes that could be used for multiple microscopy techniques would complement existing sets of tools for studying vesicle function. Here, we present a probe based on the membrane-binding C2 domain of cytosolic phospholipase A2 (cPLA2) that fulfills this need. By conjugating the C2 domain with different detectable tags, we demonstrate that a single, modular probe can allow synaptic vesicles to be imaged at multiple levels of spatial and temporal resolution. Moreover, as a general endocytic marker, the C2 domain may also be used to study membrane recycling in many cell types.

Membrane potential drives the exit from pluripotency and cell fate commitment via calcium and mTOR.

Transitioning from pluripotency to differentiated cell fates is fundamental to both embryonic development and adult tissue homeostasis. Improving our understanding of this transition would facilitate our ability to manipulate pluripotent cells into tissues for therapeutic use. Here, we show that membrane voltage (Vm) regulates the exit from pluripotency and the onset of germ layer differentiation in the embryo, a process that affects both gastrulation and left-right patterning. By examining candidate genes of congenital heart disease and heterotaxy, we identify KCNH6, a member of the ether-a-go-go class of potassium channels that hyperpolarizes the Vm and thus limits the activation of voltage gated calcium channels, lowering intracellular calcium. In pluripotent embryonic cells, depletion of kcnh6 leads to membrane depolarization, elevation of intracellular calcium levels, and the maintenance of a pluripotent state at the expense of differentiation into ectodermal and myogenic lineages. Using high-resolution temporal transcriptome analysis, we identify the gene regulatory networks downstream of membrane depolarization and calcium signaling and discover that inhibition of the mTOR pathway transitions the pluripotent cell to a differentiated fate. By manipulating Vm using a suite of tools, we establish a bioelectric pathway that regulates pluripotency in vertebrates, including human embryonic stem cells.

Development of new peptide-based tools for studying synaptic ribbon function.

Synaptic ribbons are proteinaceous specialized electron-dense presynaptic structures found in nonspiking sensory cells of the vertebrate nervous system. Understanding the function of these structures is an active area of research (reviewed in Matthews G, Fuchs P. Nat Rev Neurosci 11: 812-822, 2010). Previous work had shown that ribbons could be effectively labeled and visualized using peptides that bind to the synaptic ribbon protein RIBEYE via a PXDLS motif (Zenisek D, Horst NK, Merrifield C, Sterling P, Matthews G. J Neurosci 24: 9752-9759, 2004). Here, we expand on the previous work to develop new tools and strategies for 1) better visualizing synaptic ribbons, and 2) monitoring and manipulating calcium on the synaptic ribbon. Specifically, we developed a new higher-affinity peptide-based label for visualizing ribbons in live cells and two strategies for localizing calcium indicators to the synaptic ribbon.

Vesicle association and exocytosis at ribbon and extraribbon sites in retinal bipolar cell presynaptic terminals.

Synaptic vesicles release neurotransmitter by following a process of vesicle docking and exocytosis. Although these steps are well established, it has been difficult to observe and measure these rates directly in living synapses. Here, by combining the direct imaging of single synaptic vesicles and synaptic ribbons, I measure the properties of vesicle docking and evoked and spontaneous release from ribbon and extraribbon locations in a ribbon-type synaptic terminal, the goldfish retinal bipolar cell. In the absence of a stimulus, captured vesicles near ribbons associate tightly and only rarely undock or undergo spontaneous exocytosis. By contrast, vesicle capture at outlier sites is less stable and spontaneous exocytosis occurs at a higher rate. In response to a stimulus, exocytic events cluster near ribbons, but show no evidence of clustering away from ribbon sites. Together, the results here indicate that, although vesicles can associate and fuse both near and away from synaptic sites, vesicles at synaptic ribbons associate more stably and fusion is more tightly linked to stimuli.

Rustling synaptic vesicle cargo after exocytosis.

In this issue of Neuron, Voglmaier et al. provide new evidence that the retrieval of synaptic vesicle transporters after exocytosis proceeds along at least two different endocytic pathways. This work provides new insight into the mechanisms of sorting synaptic vesicle cargo at the cell surface.