Ribbon Synapse Formation Research Articles

GRF2 Is Crucial for Cone Photoreceptor Viability and Ribbon Synapse Formation in the Mouse Retina.

Using constitutive GRF1/2 knockout mice, we showed previously that GRF2 is a key regulator of nuclear migration in retinal cone photoreceptors. To evaluate the functional relevance of that cellular process for two putative targets of the GEF activity of GRF2 (RAC1 and CDC42), here we compared the structural and functional retinal phenotypes resulting from conditional targeting of RAC1 or CDC42 in the cone photoreceptors of constitutive GRF2KO and GRF2WT mice. We observed that single RAC1 disruption did not cause any obvious morphological or physiological changes in the retinas of GRF2WT mice, and did not modify either the phenotypic alterations previously described in the retinal photoreceptor layer of GRF2KO mice. In contrast, the single ablation of CDC42 in the cone photoreceptors of GRF2WT mice resulted in clear alterations of nuclear movement that, unlike those of the GRF2KO retinas, were not accompanied by electrophysiological defects or slow, progressive cone cell degeneration. On the other hand, the concomitant disruption of GRF2 and CDC42 in the cone photoreceptors resulted, somewhat surprisingly, in a normalized pattern of nuclear positioning/movement, similar to that physiologically observed in GRF2WT mice, along with worsened patterns of electrophysiological responses and faster rates of cell death/disappearance than those previously recorded in single GRF2KO cone cells. Interestingly, the increased rates of cone cell apoptosis/death observed in single GRF2KO and double-knockout GRF2KO/CDC42KO retinas correlated with the electron microscopic detection of significant ultrastructural alterations (flattening) of their retinal ribbon synapses that were not otherwise observed at all in single-knockout CDC42KO retinas. Our observations identify GRF2 and CDC42 (but not RAC1) as key regulators of retinal processes controlling cone photoreceptor nuclear positioning and survival, and support the notion of GRF2 loss-of-function mutations as potential drivers of cone retinal dystrophies.

Synaptic vesicle release during ribbon synapse formation of cone photoreceptors.

Mammalian cone photoreceptors enable through their sophisticated synapse the high-fidelity transfer of visual information to second-order neurons in the retina. The synapse contains a proteinaceous organelle, called the synaptic ribbon, which tethers synaptic vesicles (SVs) at the active zone (AZ) close to voltage-gated Ca2+ channels. However, the exact contribution of the synaptic ribbon to neurotransmission is not fully understood, yet. In mice, precursors to synaptic ribbons appear within photoreceptor terminals shortly after birth as free-floating spherical structures, which progressively elongate and then attach to the AZ during the following days. Here, we took advantage of the process of synaptic ribbon maturation to study their contribution to SV release. We performed whole-cell patch-clamp recordings from cone photoreceptors at three postnatal (P) development stages (P8-9, P12-13, >P30) and measured evoked SV release, SV replenishment rate, recovery from synaptic depression, domain organization of voltage-sensitive Ca2+ channels, and Ca2+-sensitivity of exocytosis. Additionally, we performed electron microscopy to determine the density of SVs at ribbon-free and ribbon-occupied AZs. Our results suggest that ribbon attachment does not organize the voltage-sensitive Ca2+ channels into nanodomains or control SV release probability. However, ribbon attachment increases SV density at the AZ, increases the pool size of readily releasable SVs available for evoked SV release, facilitates SV replenishment without changing the SV pool refilling time, and increases the Ca2+- sensitivity of glutamate release.

Disruption of Hars2 in Cochlear Hair Cells Causes Progressive Mitochondrial Dysfunction and Hearing Loss in Mice

Mutations in a number of genes encoding mitochondrial aminoacyl-tRNA synthetases lead to non-syndromic and/or syndromic sensorineural hearing loss in humans, while their cellular and physiological pathology in cochlea has rarely been investigated in vivo. In this study, we showed that histidyl-tRNA synthetase HARS2, whose deficiency is associated with Perrault syndrome 2 (PRLTS2), is robustly expressed in postnatal mouse cochlea including the outer and inner hair cells. Targeted knockout of Hars2 in mouse hair cells resulted in delayed onset (P30), rapidly progressive hearing loss similar to the PRLTS2 hearing phenotype. Significant hair cell loss was observed starting from P45 following elevated reactive oxygen species (ROS) level and activated mitochondrial apoptotic pathway. Despite of normal ribbon synapse formation, whole-cell patch clamp of the inner hair cells revealed reduced calcium influx and compromised sustained synaptic exocytosis prior to the hair cell loss at P30, consistent with the decreased supra-threshold wave I amplitudes of the auditory brainstem response. Starting from P14, increasing proportion of morphologically abnormal mitochondria was observed by transmission electron microscope, exhibiting swelling, deformation, loss of cristae and emergence of large intrinsic vacuoles that are associated with mitochondrial dysfunction. Though the mitochondrial abnormalities are more prominent in inner hair cells, it is the outer hair cells suffering more severe cell loss. Taken together, our results suggest that conditional knockout of Hars2 in mouse cochlear hair cells leads to accumulating mitochondrial dysfunction and ROS stress, triggers progressive hearing loss highlighted by hair cell synaptopathy and apoptosis, and is differentially perceived by inner and outer hair cells.

Pikachurin Is Partially Involved in the Synaptic Connection Between Donor and Host Cells in Late-Stage rd1 Mice Following Conspecific Photoreceptor Transplantation.

Photoreceptor transplantation can rescue the retinal function of late-stage rd1 mice. Many studies have used synaptic markers to suggest that there are synaptic connections after transplantation, but how donor and host cells are connected remains unknown. Many molecules are needed for triad ribbon synapse formation in wild-type mice. Among them, pikachurin is an important extracellular matrix protein that bridges the pre- and postsynaptic components. To investigate the mechanism of the synaptic connection between donor photoreceptor and host retina, we studied the expression of pikachurin in late-stage rd1 mice before and after transplantation. The results showed that the full-length form of pikachurin could still be detected in the degenerated retina. After photoreceptors were transplanted to the subretinal space of rd1 or wild-type mice, pikachurin was detected in the cytoplasm of most donor photoreceptor cells. Pikachurin puncta may represent the cleaved form of the protein and may indicate synapse generation, but it was barely observed in the donor mass of wild-type mice (3.83 ± 3.17 puncta per 100 donor cells). In contrast, pikachurin puncta could be found in the graft of the rd1 mouse retina, but the number was low (21.35 ± 9.48 puncta per 100 donor cells). In addition, 54.12 ± 8.45% of bassoon puncta were paired with pikachurin puncta and 45.5 ± 6.33% were not, indicating that there were fewer pikachurin puncta than bassoon. These results suggest that pikachurin is involved in only a portion of the synaptic connection between the donor photoreceptor and host retina.

Current concepts in cochlear ribbon synapse formation.

In mammals, hair cells and spiral ganglion neurons (SGNs) in the cochlea together are sophisticated "sensorineural" structures that transduce auditory information from the outside world into the brain. Hair cells and SGNs are joined by glutamatergic ribbon-type synapses composed of a molecular machinery rivaling in complexity the mechanoelectric transduction components found at the apical side of the hair cell. The cochlear hair cell ribbon synapse has received much attention lately because of recent and important findings related to its damage (sometimes termed "synaptopathy") as a result of noise overexposure. During development, ribbon synapses between type I SGNs and inner hair cells form in the time window between birth and hearing onset and is a process coordinated with type I SGN myelination, spontaneous activity, synaptic pruning, and innervation by efferents. In this review, we highlight new findings regarding the diversity of type I SGNs and inner hair cell synapses, and the molecular mechanisms of selective hair cell targeting. Also discussed are cell adhesion molecules and protein constituents of the ribbon synapse, and how these factors participate in ribbon synapse formation. We also note interesting new insights into the morphological development of type II SGNs, and the potential for cochlear macrophages as important players in protecting SGNs. We also address recent studies demonstrating that the structural and physiological profiles of the type I SGNs do not reach full maturity until weeks after hearing onset, suggesting a protracted development that is likely modulated by activity.

Absence of Neuroplastin-65 Affects Synaptogenesis in Mouse Inner Hair Cells and Causes Profound Hearing Loss.

In the mammalian cochlea, the sensory inner hair cells (IHCs) encode auditory information. They do this by converting sound wave-induced mechanical motion of their hair bundles into an electrical current. This current generates a receptor potential that controls release of glutamate neurotransmitter from their ribbon synapses onto the auditory afferent fiber. We show that the synapse-enriched protein Np65, encoded by the Neuroplastin gene, is localized at the IHC presynaptic region. In mutant mice, absence of Np65 causes early-onset sensorineural hearing loss and prevents normal neurotransmitter release in IHCs and colocalization of presynaptic ribbons with postsynaptic afferents. We identified Neuroplastin as a novel deafness gene required for ribbon synapse formation and function, which is critical for sound perception in mammals.

MicroRNA-200 family members are weakly expressed in the neurosensory epithelia of the developing zebrafish (Danio rerio) inner ear.

MicroRNA-200 family members are expressed in the developing mouse inner ear and in zebrafish (Danio rerio) olfactory epithelia, taste buds, and neuromasts, and have also been shown to be associated with differentiation of olfactory and taste buds. However, the role of the miR-200 family in the inner ear of zebrafish had not been studied. We investigated the expression and function of the miR-200 family in the zebrafish inner ear via in situ hybridization and loss-of-function methods. Expression of the miR-200 family was weak and dispersed throughout the developing zebrafish inner ear. After knockdown of miR-200 family members in the developing inner ear, no significant differences in development were observed compared to the controls. Otic vesicles, otoliths, and semicircular canals appeared normal. Compared with less differentiated olfactory filaments in olfactory epithelia, the development of hair cells and statoacoustic ganglion neurons were normal. The kinocilia and stereocilia of hair cells, the innervation of hair cells, and the formation of ribbon synapses were also unaffected. Overall, we conclude that the miR-200 family has a negligible role in the development of zebrafish inner ear; the functions of the miR- 200 family may be organ-specific.

Effects of mGluR6-deficiency on photoreceptor ribbon synapse formation: Comparison of electron microscopic analysis of serial sections with random sections

This study examined the effects of metabotropic glutamate receptor 6 (mGluR6) deficiency on ribbon synapse formation in rod spherules and cone pedicles using serial-section electron microscopy. In a wild-type (WT) mouse, only 3% of spherules had one invaginating bipolar dendrite (1B-type) and 97% of spherules were 2B-type. In contrast, in an mGluR6-knockout (KO) mouse, 29% of spherules were 1B-type and 71% of spherules were 2B-type. Spherules without bipolar invagination were not observed in either genotype. The single invaginating dendrites in 1B-type spherules were larger and the surface areas of synaptic ribbons were 23% smaller in the mGluR6-KO mouse than in the WT mouse. In cones, the number of invaginating bipolar dendrites decreased from 12 in the WT mouse to 9.5 in the mGluR6-KO mouse. This decrease correlated with a decrease in the number of cone synaptic ribbons from 10 in the WT mouse to 8 in the mGluR6-KO mouse. The mGluR6-KO phenotype showed negative effects on ribbon synapse formation. This negativity was similar to those in mGluR6-nob4, Gβ3-KO, Gβ5-KO, and RGS-7:RGS-11 double-KO mice, but the detailed manners and degrees of alterations appeared to vary depending on different missing components. Two published morphological assessments of the RGS-7:RGS-11 double-KO phenotype reported conflicting data; therefore, we tested the statistical techniques used in the two analyses. One statistical evaluation measure was effective in identifying a significant difference in structure between the mutant and WT phenotypes, whereas the other measure was ineffective. Conventional random section analysis using the effective measure provided sufficient data for a statistical test of the occurrence of structural changes. However, serial section analysis was required to determine the absolute numbers of ribbons and invaginating dendrites and to estimate structural parameters such as ribbon surface area.

Synaptic Ribbons Influence the Size and Frequency of Miniature-like Evoked Postsynaptic Currents

Nonspiking cells of several sensory systems respond to stimuli with graded changes in neurotransmitter release and possess specialized synaptic ribbons. Here, we show that manipulations to synaptic ribbons caused dramatic effects on mEPSC-like (mlEPSC) amplitude and frequency. Damage to rod-bipolar cell ribbons using fluorophore-assisted light inactivation resulted in the immediate reduction of mlEPSC amplitude and frequency, whereas the first evoked response after damage remained largely intact. The reduction in amplitude could not be recovered by increasing release frequency after ribbon damage. In parallel experiments, we looked at mlEPSCs from cones of hibernating ground squirrels, which exhibit dramatically smaller ribbons than awake animals. Fewer and smaller mlEPSCs were observed postsynaptic to cones from hibernating animals, although depolarized cones were able to generate larger mlEPSCs. Our results indicate that ribbon size may influence mlEPSC frequency and support a role for ribbons in coordinating multivesicular release.

Guidance-Cue Control of Horizontal Cell Morphology, Lamination, and Synapse Formation in the Mammalian Outer Retina

In the vertebrate retina, neuronal circuitry required for visual perception is organized within specific laminae. Photoreceptors convey external visual information to bipolar and horizontal cells at triad ribbon synapses established within the outer plexiform layer (OPL), initiating retinal visual processing. However, the molecular mechanisms that organize these three classes of neuronal processes within the OPL, thereby ensuring appropriate ribbon synapse formation, remain largely unknown. Here we show that mice with null mutations in Sema6A or PlexinA4 (PlexA4) exhibit a pronounced defect in OPL stratification of horizontal cell axons without any apparent deficits in bipolar cell dendrite or photoreceptor axon targeting. Furthermore, these mutant horizontal cells exhibit aberrant dendritic arborization and reduced dendritic self-avoidance within the OPL. Ultrastructural analysis shows that the horizontal cell contribution to rod ribbon synapse formation in PlexA4⁻/⁻ retinas is disrupted. These findings define molecular components required for outer retina lamination and ribbon synapse formation.

Ribeye is required for presynaptic CaV1.3a channel localization and afferent innervation of sensory hair cells

Ribbon synapses of the ear, eye and pineal gland contain a unique protein component: Ribeye. Ribeye consists of a novel aggregation domain spliced to the transcription factor CtBP2 and is one of the most abundant proteins in synaptic ribbon bodies. Although the importance of Ribeye for the function and physical integrity of ribbon synapses has been shown, a specific role in synaptogenesis has not been described. Here, we have modulated Ribeye expression in zebrafish hair cells and have examined the role of Ribeye in synapse development. Knockdown of ribeye resulted in fewer stimulus-evoked action potentials from afferent neurons and loss of presynaptic Ca(V)1.3a calcium channel clusters in hair cells. Additionally, afferent innervation of hair cells was reduced in ribeye morphants, and the reduction was correlated with depletion of Ribeye punctae. By contrast, transgenic overexpression of Ribeye resulted in Ca(V)1.3a channels colocalized with ectopic aggregates of Ribeye protein. Overexpression of Ribeye, however, was not sufficient to create ectopic synapses. These findings reveal two distinct functions of Ribeye in ribbon synapse formation--clustering Ca(V)1.3a channels at the presynapse and stabilizing contacts with afferent neurons--and suggest that Ribeye plays an organizing role in synaptogenesis.

Pikachurin, a dystroglycan ligand, is essential for photoreceptor ribbon synapse formation

Exquisitely precise synapse formation is crucial for the mammalian CNS to function correctly. Retinal photoreceptors transfer information to bipolar and horizontal cells at a specialized synapse, the ribbon synapse. We identified pikachurin, an extracellular matrix-like retinal protein, and observed that it localized to the synaptic cleft in the photoreceptor ribbon synapse. Pikachurin null-mutant mice showed improper apposition of the bipolar cell dendritic tips to the photoreceptor ribbon synapses, resulting in alterations in synaptic signal transmission and visual function. Pikachurin colocalized with both dystrophin and dystroglycan at the ribbon synapses. Furthermore, we observed direct biochemical interactions between pikachurin and dystroglycan. Together, our results identify pikachurin as a dystroglycan-interacting protein and demonstrate that it has an essential role in the precise interactions between the photoreceptor ribbon synapse and the bipolar dendrites. This may also advance our understanding of the molecular mechanisms underlying the retinal electrophysiological abnormalities observed in muscular dystrophy patients.

Synaptic organization in cochlear inner hair cells deficient for the CaV1.3 (α1D) subunit of L-type Ca2+ channels

Cochlear inner hair cells (IHCs) release neurotransmitter onto afferent auditory nerve fibers in response to sound stimulation. Normal development and function of inner hair cells require the expression of α subunit 1.3 forming L-type voltage-gated Ca2+ channel (CaV1.3).Here, we used immunohistochemistry and reverse transcription-polymerase chain reaction to study the synaptic organization and expression of large conductance Ca2+-activated potassium channels in IHCs of mice lacking the CaV1.3 Ca2+ channel (CaV1.3−/−). Despite the near complete block of evoked afferent synaptic transmission, hair cell ribbon synapses were formed and remained preserved for at least 4 weeks after birth. Moreover, these “silent” afferent synapses held major components of the synaptic machinery such as Bassoon, Piccolo, and CSP. Hence, the block of exocytosis might be solely attributed to the lack of Ca2+ influx through CaV1.3 channels. Later on, CaV1.3 deficient IHCs subsequently lost their afferent synapses. This was probably due to a secondary degeneration of the postsynaptic spiral ganglion neurons.In line with a prolonged efferent synaptic transmission onto CaV1.3 deficient IHCs, which normally ceases around onset of hearing, we found juxtaposed immunoreactive spots of efferent presynaptic synaptophysin and postsynaptic (IHCs) small conductance Ca2+-activated potassium channels (SK channels) up to six weeks after birth. Finally, we show a substantial reduction of mRNA for the α subunit of the large conductance Ca2+-activated potassium channel (BK) in the apical cochlea, suggesting a reduced transcription of its gene in CaV1.3 deficient IHCs. CaV1.3 deficient IHCs lacked the apical spot-like immunoreactivity of clustered BK channels, which normally contribute to the temporal precision of hair cell afferent synaptic transmission.In summary, these data indicate that the CaV1.3 channels are crucially involved in regulation of the expression of BK and SK channels. CaV1.3 channels seem not to be essential for ribbon synapse formation, but are required for the maintenance of ribbon synapses and spiral ganglion neurons.

Expression of vesicular glutamate transporter 1 in the mouse retina reveals temporal ordering in development of rod vs. cone and ON vs. OFF circuits.

Glutamatergic transmission is crucial to the segregation of ON and OFF pathways in the developing retina. The temporal sequence of maturation of vesicular glutamatergic transmission in rod and cone photoreceptor and ON and OFF bipolar cell terminals is currently unknown. Vesicular glutamate transporters (VGLUTs) that load glutamate into synaptic vesicles are necessary for vesicular glutamatergic transmission. To understand better the formation and maturation of glutamatergic transmission in the rod vs. cone and ON vs. OFF pathways of the retina, we examined the developmental expression of VGLUT1 and VGLUT2 immunocytochemically in the mouse retina. Photoreceptor and bipolar cell terminals showed only VGLUT1-immunoreactivity (-IR); no VGLUT2-IR was present in any synapses of the developing or adult retina. VGLUT1-IR was first detected in cone photoreceptor terminals at postnatal day 2 (P2), several days before initiation of ribbon synapse formation at P4-P5. Rod terminals showed VGLUT1-IR by P8, when they invade the outer plexiform layer (OPL) and initiate synaptogenesis. Developing OFF bipolar cell terminals showed VGLUT1-IR around P8, 2-3 days after bipolar terminals were first identified in the inner plexiform layer (IPL) by labeling for the photoreceptor and bipolar cell terminal marker, synaptic vesicle protein 2B. Although terminals of ON bipolar cells were present in the IPL by P6-P8, most did not show VGLUT1-IR until P8-P10 and increased dramatically from P12. These data suggest a hierarchical development of glutamatergic transmission in which cone circuits form prior to rod circuits in both the OPL and IPL, and OFF circuits form prior to ON circuits in the IPL.

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.

The expression pattern and assembly profile of synaptic membrane proteins in ribbon synapses of the developing mouse retina.

In the present study, we generated a systematic overview of the expression pattern and assembly profile of synaptic membrane proteins in ribbon synapses of the developing mouse retina. Using indirect immunofluorescence microscopy, we analyzed the spatial and temporal distribution of 11 important membrane and membrane-associated synaptic proteins (syntaxin 1/3, SNAP-25, synaptobrevin 2, synaptogyrin, synaptotagmin I, SV2A, SV2B, Rab3A, clathrin light chains, CSP and neuroligin I) during synaptogenesis. The temporospatial distribution of these synaptic proteins was "normalized" by the simultaneous visualization of the synaptic vesicle protein synaptophysin, which served as an internal reference protein. We found that expression of various synaptic membrane proteins started at different time points and changed progressively during development. At early stages of development synaptic vesicle membrane proteins at extrasynaptic locations did not always colocalize with synaptophysin, indicating that these proteins probably do not reside in the same transport vesicles. Despite a non-synchronized onset of protein expression, clustering and colocalization of all synaptic membrane proteins at ribbon synapses roughly occurred in the same time window (between day 4 after birth, P4, and P5). Thus, the basic synaptic membrane machinery is already present in ribbon synapses before the well-known complete morphological maturation of ribbon synapses between P7 and P12. We conclude that ribbon synapse formation is a multistep process in which the concerted recruitment of synaptic membrane proteins is a relatively early event and clearly not the final step.

Vesicular Neurotransmitter Transporter Expression in Developing Postnatal Rodent Retina: GABA and Glycine Precede Glutamate

Vesicular transporters regulate the amount and type of neurotransmitter sequestered into synaptic vesicles and, hence, the kind of signal transmitted to postsynaptic neurons. Glutamate is the prominent excitatory neurotransmitter in retina; GABA and glycine are the main inhibitory neurotransmitters. Little is known about the ontogeny of vesicular neurotransmission in retina. We investigated expression of glutamatergic [vesicular glutamate transporter 1 (VGLUT1)] and GABA/glycinergic [vesicular GABA/glycine transporter (VGAT)] vesicular transporters in postnatal retina. VGLUT1 labels glutamatergic synapses. VGLUT1 and synaptic vesicle 2 colocalized to photoreceptor terminals. VGLUT1 colocalized with PKC to rod bipolar terminals and to ON bipolar terminals in metabotropic glutamate receptor 6+/- mice. Developmentally, VGAT expression precedes VGLUT1. In rat and mouse retina, VGAT occurred in the inner retina by postnatal day 1 (P1). In rat retina, VGLUT1 was in the outer retina by P5-P7 and the inner retina by P7. In the mouse retina, VGLUT1 expression was in the outer retina by P3 and the inner retina by P5. Both rat and mouse retina had an adult pattern of VGLUT1 expression by P14. VGLUT1 expression precedes ribbon synapses, which are first observed in the inner retina at P11 (Fisher, 1979) in mouse and P13 (Horsburgh and Sefton, 1987) in rat. The ribbon synapse marker RIBEYE was not detected in inner retina of P5 or P7 rat. Spontaneous EPSCs in mouse ganglion cells were recorded as early as P7. Together, these findings indicate that vesicular GABA and glycine transmission precedes vesicular glutamate transmission in developing rodent retina. Furthermore, vesicular glutamate transmission likely occurs before ribbon synapse formation in the inner retina.

Histological and electron microscopic milestones in the development of the retina of a marsupial wallaby, Macropus eugenii.

Retinal differentiation in the pouch young of the wallaby Macropus eugenii was characterized microscopically and morphometrically. Mitosis occurs until early in the second month in the central retina, and until early in the fourth month, peripherally. Separation of the neuroblast layer by the outer plexiform layer did not immediately halt cell division. The retinal surface continued to expand well past the time of cessation of proliferation. Cell death in the ganglion cell layer continued through the fourth month centrally and to nearly five months in the periphery. The major period of cell death was coincident with the segregation of retinal afferents and the refinement of topography in the superior colliculus and dorsal lateral geniculate nucleus. Beginning in the third month retinal thickness, measured between the outer limiting membrane and nerve fiber layer, declined equally in peripheral and central regions. At all stages the combined thicknesses of the outer and inner nuclear layer in the retinal periphery was greater than that in the center. Together with a late thickening of the inner plexiform layer, the data are consistent with the suggestion that expansion of peripheral non-ganglion cell elements may play a role in development of center to periphery differences in ganglion cell distributions. Retinal differentiation of the wallaby follows the pattern of most mammals. The onset of development of key milestones for the acquisition of retinal function occurred in the sequence: conventional synapse formation prior to ribbon synapse formation in the inner plexiform layer, and photoreceptor outer segment differentiation prior to terminal triad synapse formation.

Synaptogenesis in the retina of the cat

We have studied the development of synapses in the retina of the cat from E(embryonic day)21 to adulthood. The inner plexiform layer (IPL) could be distinguished by E36, but at this age no synapses had formed, although compact processes had formed in the IPL and membrane specialisations had developed in adjacent processes. Conventional synapses form in the IPL from E45 and become increasingly numerous and differentiated over subsequent weeks. Extracellular space and cellular debris were prominent during the formation of these synapses. The conventional synapses appear to form principally between amacrine cells until E56, when ganglion cell dendrites could be identified as postsynaptic processes. Ribbon synapses characteristic of bipolar cells were identified around birth, suggesting that bipolar cells do not form synapses until that age. The outer plexiform layer (OPL) could be distinguished in central retina at E56. Extracellular space, debris of degenerating cells and mounds of agranular vesicles were prominent at this age but synapses were not observed until E59, when cone pedicles formed ribbon synapses onto horizontal cell processes. The first synapses clearly formed by spherules, also onto horizontal cells, were seen at E62. The central process of the postsynaptic triad, considered to be the dendrite of a bipolar cell, was first observed in both cone pedicles and rod spherules around birth, again suggesting that bipolar cells do not enter into synaptic arrangements until that age. Synaptogenesis in the OPL shows a strong centro-peripheral gradient; its initial stages were observed centrally in the late E50's but synapse formation was not complete in the retinal periphery until P(postnatal day)7 or later. We could not detect a centro-peripheral gradient in the formation of conventional synapses in the IPL, but the formation of ribbon synapses in this layer began centrally at birth and in the mid-periphery at P5. In summary, the first synapses to form in the retina are those which spread information laterally within the plexiform layers, between amacrine cells and from receptor to horizontal cells. The cells which carry information centrally, in particular bipolar cells, enter into synaptic arrangements considerably later. Further, retinal cells seem to form synapses in a distinct sequence: first amacrines, then receptors and lastly bipolar cells.

A freeze-fracture study of photoreceptor presynaptic membranes during ribbon synapse formation.

The outer plexiform layer (OPL) of the developing chick retina from 11 embryonic days to 11/2 weeks posthatching has been studied by freeze-fracture to characterize changes in the membrane structure of photoreceptor terminals during synaptogenesis. At early stages, the undifferentiated photoreceptor synaptic base is characterized by a sparse distribution of intramembrane articles on the inner leaflet (P-face). Later, as the synaptic base begins to differentiate by extending filopodia into the OPL, numerous small aggregates of large particles appear between and on filopodial surfaces. Many of the aggregates occupy crater-like expressions, which are seen in cross-fractures through the underlying cytoplasm to be associated with vesicular invaginations of the presynaptic membrane. Corresponding thin sections through these regions at this time reveal immature arciform densities and coated vesicles fusing with the presynaptic membrane adjacent to these densities. At later stages, many of the particle aggregates on the photoreceptor membrane appear to have coalesced into longer arrays overlying ridges surrounded by numerous vesicle fusion sites. These intramembrane changes correlate with the formation of the mature arciform density-synaptic ribbon specialization in the photoreceptor presynaptic terminal and with physiological maturation of the chick retina.