Overexpression of guanylate cyclase activating protein 2 in rod photoreceptors in vivo leads to morphological changes at the synaptic ribbon

Guanylate cyclase activating proteins are EF-hand containing proteins that confer calcium sensitivity to retinal guanylate cyclase at the outer segment discs of photoreceptor cells. By making the rate of cGMP synthesis dependent on the free intracellular calcium levels set by illumination, GCAPs play a fundamental role in the recovery of the light response and light adaptation. The main isoforms GCAP1 and GCAP2 also localize to the synaptic terminal, where their function is not known. Based on the reported interaction of GCAP2 with Ribeye, the major component of synaptic ribbons, it was proposed that GCAP2 could mediate the synaptic ribbon dynamic changes that happen in response to light. We here present a thorough ultrastructural analysis of rod synaptic terminals in loss-of-function (GCAP1/GCAP2 double knockout) and gain-of-function (transgenic overexpression) mouse models of GCAP2. Rod synaptic ribbons in GCAPs-/- mice did not differ from wildtype ribbons when mice were raised in constant darkness, indicating that GCAPs are not required for ribbon early assembly or maturation. Transgenic overexpression of GCAP2 in rods led to a shortening of synaptic ribbons, and to a higher than normal percentage of club-shaped and spherical ribbon morphologies. Restoration of GCAP2 expression in the GCAPs-/- background (GCAP2 expression in the absence of endogenous GCAP1) had the striking result of shortening ribbon length to a much higher degree than overexpression of GCAP2 in the wildtype background, as well as reducing the thickness of the outer plexiform layer without affecting the number of rod photoreceptor cells. These results indicate that preservation of the GCAP1 to GCAP2 relative levels is relevant for maintaining the integrity of the synaptic terminal. Our demonstration of GCAP2 immunolocalization at synaptic ribbons at the ultrastructural level would support a role of GCAPs at mediating the effect of light on morphological remodeling changes of synaptic ribbons.     

An ultrastructural study of embryonic chick retinal neurons in culture

Summary The differentiation of cells and synapses in explants of 9-day-old chick embryo retina has been studied by light and electron microscopy over a period of 35 days in vitro, and samples of retina from the 9-day chick foetus were directly fixed and prepared for study.At the time of explantation the retinae were poorly differentiated and no lamination was apparent. From day 14 onwards, (i) outer and inner nuclear layers (ONL, INL) separated by a layer of neuropil corresponding to the outer plexiform layer (OPL) and (ii) a layer of scattered large ganglion cells separated from the INL by a zone of neuropil resembling the inner plexiform layer (IPL) were apparent, and (iii) a well-differentiated outer limiting membrane was established close to the surface of the explants. In the oldest cultures some development of photoreceptor outer segments occurred but a distinct optic nerve fibre layer did not form.Although cell identification presented problems even in the oldest cultures, the major retinal cell types described in vivo could be identified. Photoreceptor cells developed pedicles in the OPL which became filled with synaptic vesicles and synaptic ribbons and established ribbon synapses (including triads) with and were commonly invaginated by processes from horizontal and bipolar cells. Processes of bipolar cells in the IPL formed simple and dyad synapses. At least two types of presynaptic amacrine cells were also identified in the INL, one of which contained large numbers of dense-core vesicles. The ganglion cells, though sparse, were large and well differentiated.These findings show that all the major neuronal types of the retina are capable of developing and differentiating in vitro, lagging behind the time-table of development and differentiation in vivo by approximately 7 days, but resulting in a histotypically organised retina with synaptic neuropil showing many similarities to the corresponding neuropil in vivo.     

How to make a synaptic ribbon: RIBEYE deletion abolishes ribbons in retinal synapses and disrupts neurotransmitter release

Synaptic ribbons are large proteinaceous scaffolds at the active zone of ribbon synapses that are specialized for rapid sustained synaptic vesicles exocytosis. A single ribbon‐specific protein is known, RIBEYE, suggesting that ribbons may be constructed from RIBEYE protein. RIBEYE knockdown in zebrafish, however, only reduced but did not eliminate ribbons, indicating a more ancillary role. Here, we show in mice that full deletion of RIBEYE abolishes all presynaptic ribbons in retina synapses. Using paired recordings in acute retina slices, we demonstrate that deletion of RIBEYE severely impaired fast and sustained neurotransmitter release at bipolar neuron/AII amacrine cell synapses and rendered spontaneous miniature release sensitive to the slow Ca²⁺‐buffer EGTA, suggesting that synaptic ribbons mediate nano‐domain coupling of Ca²⁺ channels to synaptic vesicle exocytosis. Our results show that RIBEYE is essential for synaptic ribbons as such, and may organize presynaptic nano‐domains that position release‐ready synaptic vesicles adjacent to Ca²⁺ channels.     

The presynaptic active zone protein bassoon is essential for photoreceptor ribbon synapse formation in the retina

The photoreceptor ribbon synapse is a highly specialized glutamatergic synapse designed for the continuous flow of synaptic vesicles to the neurotransmitter release site. The molecular mechanisms underlying ribbon synapse formation are poorly understood. We have investigated the role of the presynaptic cytomatrix protein Bassoon, a major component of the photoreceptor ribbon, in a mouse retina deficient of functional Bassoon protein. Photoreceptor ribbons lacking Bassoon are not anchored to the presynaptic active zones. This results in an impaired photoreceptor synaptic transmission, an abnormal dendritic branching of neurons postsynaptic to photoreceptors, and the formation of ectopic synapses. These findings suggest a critical role of Bassoon in the formation and the function of photoreceptor ribbon synapses of the mammalian retina.     

Differential expression of syntaxin-1 and synaptophysin in the developing and adult human retina

Synaptophysin and syntaxin-1 are membrane proteins that associate with synaptic vesicles and presynaptic active zones at nerve endings, respectively. The former is known to be a good marker of synaptogenesis; this aspect, however, is not clear with syntaxin-1. In this study, the expression of both proteins was examined in the developing human retina and compared with their distribution in postnatal to adult retinas, by immunohistochemistry. In the inner plexiform layer, both were expressed simultaneously at 11–12 weeks of gestation, when synaptogenesis reportedly begins in the central retina. In the outer plexiform layer, however, the immunoreactivities were prominent by 16 weeks of gestation. Their expression in both plexiform layers followed a centre-to-periphery gradient. The immunoreactivities for both proteins were found in the immature photoreceptor, amacrine and ganglion cells; however, synaptophysin was differentially localized in bipolar cells and their axons, and syntaxin was present in some horizontal cells. In postnatal-to-adult retinas, synaptophysin immunoreactivity was prominent in photoreceptor terminals lying in the outer plexiform layer; on the contrary, syntaxin-1 was present in a thin immunoreactive band in this layer. In the inner plexiform layer, however, both were homogeneously distributed. Our study suggests that (i) syntaxin-1 appears in parallel with synapse formation; (ii) synaptogenesis in the human retina might follow a centre-to-periphery gradient; (iii) syntaxin-1 is likely to be absent from ribbon synapses of the outer plexiform layer, but may occur at presynaptic terminals of photoreceptor and horizontal cells, as is apparent from its localization in these cells, which is hitherto unreported for any vertebrate retina.     

Ultrastructure of the synaptic ribbons in photoreceptor cells of Rana catesbeiana revealed by freeze-etching and freeze-substitution

Summary The three-dimensional structure of synaptic ribbons in photoreceptor cells of the frog retina was studied with freeze-etching and freeze-substitution methods, combined with a rapid-freezing technique. Although the synaptic ribbon consisted of two electron-dense plaques bisected by an electron-lucent layer in conventional thin sections, such lamellar nature was not so evident in freeze-etched replicas. The cytoplasmic surfaces of the synaptic ribbon presented an extremely regular arrangements of small particles 4–6 nm in diameter. Fine filaments 8–10 nm in diameter and 30–50 nm in length connected synaptic vesicles and the ribbon surface. These connections were mediated by large particles on both ends of the filaments. Approximately 3–5 filaments attached to one synaptic vesicle. Synaptic ribbons were anchored to a characteristic meshwork underlying the presynaptic membrane via another group of similar fine filaments. The meshwork seemed to be an etched replicated image of the presynaptic archiform density observed in thin sections.     

Fine structure of the retina of black bass, Micropterus salmoides (Centrarchidae, Teleostei).

The structure of light- and dark-adapted retina of the black bass, Micropterus salmoides has been studied by light and electron microscopy. This retina lacks blood vessels at all levels. The optic fiber layer is divided into fascicles by the processes of Müller cells and the ganglion cell layer is represented by a single row of voluminous cells. The inner nuclear layer consists of two layers of horizontal cells and bipolar, amacrine and interplexiform cells. In the outer plexiform layer we observed the synaptic terminals of photoreceptor cells, rod spherules and cone pedicles and terminal processes of bipolar and horizontal cells. The spherules have a single synaptic ribbon and the pedicles possess multiple synaptic ribbons. Morphologically, we have identified three types of photoreceptors: rods, single cones and equal double cones which undergo retinomotor movements in response to changes in light conditions. The cones are arranged in a square mosaic whereas the rods are dispersed between the cones.     

Localization of vanilloid receptor 1 (TRPV1/VR1)-like immunoreactivity in goldfish and zebrafish retinas: Restriction to photoreceptor synaptic ribbons

The vanilloid receptor type 1 (TRPV1/VR1) is a non-specific calcium-permeable ionotropic cation channel expressed in the peripheral sensory system as well as in the central nervous system. An endogenous ligand for TRPV1 is arachidonoyl ethanolamide (anandamide), which also activates the metabotropic cannabinoid receptor 1 (CB1). Previous studies in this laboratory reported CB1 receptors and CB1-mediated effects on voltage-gated currents in goldfish cones and bipolar cells. In this study, we show TRPV1-like-immunoreactivity (TRPV1-L-IR) by immunoblot analysis of goldfish retina and rat brain homogenates with a guinea pig polyclonal antibody against the C-terminus of the rat TRPV1. Light-level immunocytochemistry showed restriction of the guinea pig-TRPV1 antibody to a very narrow band in the outer plexiform layer in goldfish and zebrafish retinas. However, no immunoreactivity was detected using rabbit-polyclonal antibodies against the C or N-termini of the rat TRPV1. Pre and post-embedding electron microscopy (EM) immunocytochemistry revealed that TRPV1-L-IR (using the guinea pig antibody) was restricted to synaptic ribbons of all cones and many rods, but never was observed at the synaptic ribbons of bipolar cells. While pre-embedded tissue showed diffuse label associated only with photoreceptor-synaptic ribbons, analysis of post-embedded tissue showed label tightly restricted to synaptic ribbons of all cones and many rods. Oblique sections showed that immunogold particles were confined to the outer electron dense region of the ribbons, with few or no gold particles in the ribbon core or associated with tethers or vesicles. Although TRPV1-L-IR described here, does not necessarily represent TRPV1 antigen associated with synaptic ribbons, these data provide an unequivocal label with which to study the functional dynamics of ribbon formation and degradation in teleost photoreceptors.     

Rod cells dissociated from mature salamander retina: ultrastructure and uptake of horseradish peroxidase

To test the effects of isolation on adult neurons, we investigated the fine structure and synaptic activity of rod cells dissociated from the mature salamander retina and maintained in vitro. First, freshly isolated rod cells appeared remarkably similar to their counterparts in the intact retina: the outer segment retained its stack of membranous disks and the inner segment contained its normal complements of organelles. Some reorganization of the cell surface, however, was observed: (a) radial fins, present at the level of the cell body, were lost; and (b) the apical and distal surfaces of the inner and outer segments, respectively became broadly fused. Second, the synaptic endings or pedicles retained their presynaptic active zones: reconstruction of serially sectioned pedicles by using three-dimensional computer graphics revealed that 73% of the synaptic ribbons remained attached to the plasmalemma either at the cell surface or along its invaginations. Finally, tracer experiments that used horseradish peroxidase demonstrated that dissociated rod cells recycled synaptic vesicle membrane in the dark and thus probably released transmitter by exocytosis. Under optimal conditions, a maximum of 40% of the synaptic vesicles within the pedicle were labeled. As in the intact retina, uptake of horseradish peroxidase was suppressed by light. Thus, freshly dissociated receptor neurons retained many of their adult morphological and physiological characteristics. In long-term culture, the photoreceptors tended to round up; however, active zones were present even 2 wk after removal of the postsynaptic processes.     

Diurnal changes in synaptic ribbons of rod cells of the turtle

Diurnal morphological changes in synaptic ribbons of the rod cells of the turtle were revealed by electron microscopy with serial ultrathin sections. Freshwater turtles (Pseudemys) were maintained under the light-dark cycle with lights on from 0600 to 1800 hr. Retinas around the posterior pole of the eyeball were fixed in 2.5% glutaraldehyde and 1% osmium tetroxide. In total, 124 rod cells and several hundred cone cells were observed. At 0000 (midnight), ribbons are situated close to and perpendicular to the presynaptic membrane. They show single stick-shaped profiles on thin section. From midnight toward noon, the stick-shaped ribbons grow into large multilayered ribbon complexes composed of several sticks arranged parallel to each other. Then, the ribbon complexes begin to disintegrate into irregular fragments from noon toward night. At 1900, aggregates of rounded or club-shaped ribbons are predominant.     

The ultrastructural differentiation of synaptic sites in the inner plexiform layer of a teleostean retina.

The differentiation of axon terminals in the retina inner plexiform layer was studied by electron microscopy, with special reference to synaptic junctions, number and size of synaptic vesicles, dense core vesicles, biogenic amines and ATPase. Five types of synaptic junctions were found, including a ribbon type. They appear on different days of embryonic life and show different patterns of increase. The ultrastructural differentiation of the most frequent type is described in detail. The numbers of synaptic vesicles indicate the existence of three types of axon terminals which appear on different days. These and other data lead to the following model: The contacts between amacrine cells are the first to appear, followed by amacrine--bipolar cell contacts and amacrine--ganglion cell contacts. The latter are the most frequent ones and increase immediately after having appeared, while amacrine--bipolar cell contacts increase only some days later, which is also the case for bipolar--amacrine cell contacts. Biogenic amines and cytochemically detectable ATPase appear along with the formation of synaptic sites.     

An ethanolic phosphotungstic acid (EPTA) analysis of photoreceptor and synaptic ultrastructure in the guinea pig retina

Ethanolic phosphotungstic acid (EPTA) has been used to elucidate the structure of certain organelles contained within retinal cells not clearly discernible using conventional preparations. Both synaptic and nonsynaptic components of the guinea pig neural retina have been analyzed. Within the photoreceptor (PR) cell EPTA-stained components include the connecting cilia, their basal bodies, and the root filament system. Cross-striated fibrillar organelles, similar in appearance to the root filaments, are also observed in the nuclear region, the synaptic terminal and other parts of the PR cell. The possible structural continuity and significance of these structures are discussed. Within retinal synapses of both the inner and outer plexiform layers, ribbons and associated paramembranous specializations are stained. The photoreceptor ribbons have a trialaminar structure with filamentous, tufted borders. Synaptic cleft material and postsynaptic densities are also stained. Bipolar cell synapses in the inner plexiform layer contain stained short ribbons as well as closely associated peg-like densities extending towards the presynaptic membrane.     

Neurotransmitter release at ribbon synapses in the retina

The synapses of photoreceptors and bipolar cells in the retina are easily identified ultrastructurally by the presence of synaptic ribbons, electron-dense bars perpendicular to the plasma membrane at the active zones, extending about 0.5 microm into the cytoplasm. The neurotransmitter, glutamate, is released continuously (tonically) from these 'ribbon synapses' and the rate of release is modulated in response to graded changes in the membrane potential. This contrasts with action potential-driven bursts of release at conventional synapses. Similar to other synapses, neurotransmitter is released at ribbon synapses by the calcium-dependent exocytosis of synaptic vesicles. Most components of the molecular machinery governing transmitter release are conserved between ribbon and conventional synapses, but a few differences have been identified that may be important determinants of tonic transmitter release. For example, the presynaptic calcium channels of bipolar cells and photoreceptors are different from those elsewhere in the brain. Differences have also been found in the proteins involved in synaptic vesicle recruitment to the active zone and in synaptic vesicle fusion. These differences and others are discussed in terms of their implications for neurotransmitter release from photoreceptors and bipolar cells in the retina.     

Localization of metabotropic glutamate receptors in the outer plexiform layer of the goldfish retina

We studied the localization of metabotropic glutamate receptors (mGluRs) in the goldfish outer plexiform layer by light-and electron-microscopical immunohistochemistry. The mGluR1α antibody labeled putative ON-type bipolar cell dendrites and horizontal cell processes in both rod spherules and cone triads. Immunolabeling for mGluR2/3 was absent in the rod synaptic complex but was found at horizontal cell dendrites directly opposing the cone synaptic ribbon. The mGluR5 antibody labeled Müller cell processes wrapping rod terminals and horizontal cell somata. The mGluR7 antibody labeled mainly horizontal cell dendrites invaginating rods and cones and some putative bipolar cell dendrites in the cone synaptic complex. The finding of abundant expression of various mGluRs in bipolar and horizontal cell dendrites suggests multiple sites of glutamatergic modulation in the outer retina. Financial support for this work was provided by Conselho Nacional de Pesquisa (CNPq), Brazil (grant 200915/98-3 to C.J.)     

Ultrastructure of the distal retina of the adult zebrafish, Danio rerio

The organization, morphological characteristics, and synaptic structure of photoreceptors in the adult zebrafish retina were studied using light and electron microscopy. Adult photoreceptors show a typical ordered tier arrangement with rods easily distinguished from cones based on outer segment (OS) morphology. Both rods and cones contain mitochondria within the inner segments (IS), including the large, electron-dense megamitochondria previously described (Kim et al.) Four major ultrastructural differences were observed between zebrafish rods and cones: (1) the membranes of cone lamellar disks showed a wider variety of relationships to the plasma membrane than those of rods, (2) cone pedicles typically had multiple synaptic ribbons, while rod spherules had 1-2 ribbons, (3) synaptic ribbons in rod spherules were ∼2 times longer than ribbons in cone pedicles, and (4) rod spherules had a more electron-dense cytoplasm than cone pedicles. Examination of photoreceptor terminals identified four synaptic relationships at cone pedicles: (1) invaginating contacts postsynaptic to cone ribbons forming dyad, triad, and quadrad synapses, (2) presumed gap junctions connecting adjacent postsynaptic processes invaginating into cone terminals, (3) basal junctions away from synaptic ribbons, and (4) gap junctions between adjacent photoreceptor terminals. More vitread and slightly farther removed from photoreceptor terminals, extracellular microtubule-like structures were identified in association with presumed horizontal cell processes in the OPL. These findings, the first to document the ultrastructure of the distal retina in adult zebrafish, indicate that zebrafish photoreceptors have many characteristics similar to other species, further supporting the use of zebrafish as a model for the vertebrate visual system.     

Transient synaptic ribbons in the mammalian retina at unusual sites

In the vertebrate retina the presence of synaptic ribbons (SRs) is well documented in two sites only, viz., in photoreceptor axon terminals in the outer plexiform layer and in bipolar cell axons in the inner plexiform layer. The present paper reports the presence of non-photoreceptor SRs in the outer plexiform layer of cattle and mouse, where they were seen in small numbers in thin cell processes near cone pedicles of light-adapted animals. They were never seen near rod spherules. Quantitative data obtained in mice killed at different time-points revealed that the SRs under consideration increased in number during day time and were absent during the dark phase. Moreover, under high light intensity of 10000 lux they were more frequent in number compared to 100-lux-exposed animals. It is concluded that the cell processes revealing the temporary presence of SRs are processes of flat bipolar cells which may provide a feedback to cones during the light phase.     

The Molecular Architecture of Ribbon Presynaptic Terminals

The primary receptor neurons of the auditory, vestibular, and visual systems encode a broad range of sensory information by modulating the tonic release of the neurotransmitter glutamate in response to graded changes in membrane potential. The output synapses of these neurons are marked by structures called synaptic ribbons, which tether a pool of releasable synaptic vesicles at the active zone where glutamate release occurs in response to calcium influx through L-type channels. Ribbons are composed primarily of the protein, RIBEYE, which is unique to ribbon synapses, but cytomatrix proteins that regulate the vesicle cycle in conventional terminals, such as Piccolo and Bassoon, also are found at ribbons. Conventional and ribbon terminals differ, however, in the size, molecular composition, and mobilization of their synaptic vesicle pools. Calcium-binding proteins and plasma membrane calcium pumps, together with endomembrane pumps and channels, play important roles in calcium handling at ribbon synapses. Taken together, emerging evidence suggests that several molecular and cellular specializations work in concert to support the sustained exocytosis of glutamate that is a hallmark of ribbon synapses. Consistent with its functional importance, abnormalities in a variety of functional aspects of the ribbon presynaptic terminal underlie several forms of auditory neuropathy and retinopathy.     

Differential sensitivity of rods and cones in Xenopus retina to hemicholinium-3

Summary Hemicholinium-3 (HC-3), a drug which prevents synthesis of acetylcholine in neurons, when injected intraperitoneally in doses as low as 2×5 mg/kg produces marked ultrastructural changes and damage in rod but not in cone photoreceptors. In rods it causes a reduction in cytoplasmic back-ground density, swelling of the endoplasmic reticulum, ballooning of the outer membrane of the nucleus, leaching of the nucleoplasm and clumping of the nuclear chromatin. In dark-adapted rods HC-3 produces some loss of cytoplasmic synaptic vesicles but no reduction in numbers of those vesicles which lie adjacent to the synaptic ribbons. In light-adapted rods the drug does not cause such an apparent reduction of synaptic vesicles but does induce a considerable reduction in numbers of vesicles associated with the ribbons. These structural changes are discussed in the light of what is known about the pharmacology of HC-3 and neurotransmitter release from vertebrate photoreceptors.The authors wish to thank SRC for a grant in support of this work     

Structure suggests function: the case for synaptic ribbons as exocytotic nanomachines

Synaptic ribbons, the organelles identified in electron micrographs of the sensory synapses involved in vision, hearing, and balance, have long been hypothesized to play an important role in regulating presynaptic function because they associate with synaptic vesicles at the active zone. Their physiology and molecular composition have, however, remained largely unknown. Recently, a series of elegant studies spurred by technical innovation have finally begun to shed light on the ultrastructure and function of ribbon synapses. Electrical capacitance measurements have provided sub-millisecond resolution of exocytosis, evanescent-wave microscopy has filmed the fusion of single 30 nm synaptic vesicles, electron tomography has revealed the 3D architecture of the synapse, and molecular cloning has begun to identify the proteins that make up ribbons. These results are consistent with the ribbon serving as a vesicle "conveyor belt" to resupply the active zone, and with the suggestion that ribbon and conventional chemical synapses have much in common.