Formation of synaptic specializations in the inner plexiform layer of the developing chick retina

The development of synapse-like specializations was investigated in the inner plexiform layer of the developing chick retina by using light and electron microscopy. Six monoclonal antibodies, directed against glycine and γ-aminobutyric acid (GABA)_A receptor subunits, the intracellular receptor-associated protein gephyrin, synaptotagmin, and synaptophysin were used to determine the initial appearance and distribution of their antigens. Synaptophysin and synaptotagmin immunoreactivity was detected in the retina concurrent with the formation of the inner plexiform layer at embryonic day 7. This early appearance before synaptic differentiation, together with the transient expression of synaptotagmin immunoreactivity in the synapse-free optic fiber layer, suggests that in the developing central nervous system (CNS) these proteins are not confined to synapses. The first immunofluorescence signal detected with specific antibodies against the β₂ and β₃-subunits of the GABA_A receptor, the glycine receptor, and gephyrin appeared at embryonic day 12. In contrast, the α₁-subunit of the adult-type glycine receptor heteromeric complex was detectable only at later stages of development, after embryonic day 16, suggesting a change in the subunit composition of some glycine receptor complexes. The staining was clearly punctate, indicating the clustering of the α₁-subunit at synapses. Electron microscopic investigation revealed the first postsynaptic densities and active zones in the inner plexiform layer of the retina at embryonic day 12. These results reveal different patterns of development for the investigated pre- and postsynaptic proteins and indicate a parallel appearance of gephyrin, glycine receptor, and the β₂ and β₃-subunits of the GABA_A receptor with the first synaptic specializations in the inner plexiform layer of the developing chick retina. © 1996 Wiley-Liss, Inc.     

Development of synaptic arrays in the inner plexiform layer of neonatal mouse retina.

Retinas from mice of the C57BL/6 strain were sampled at frequent intervals from birth to postnatal day 33 to determine the numerical density of conventional and ribbon synapses within the inner plexiform layer (IPL) as a function of time. Synaptic arrays of the IPL were formed in three phases. During Phase I, from day 3 to day 10, conventional synapses were produced at a mean rate of 0.44 synapses/1,000 micrometer3/hour, but no ribbons were seen. During Phase II, from day 11 to day 15, ribbons formed at a rate of 0.38 ribbons/1,000 micrometer3/hour and conventional synapses were produced at a rate of 1.15 synapses/1,000 micrometer3/hour. Phase III began at day 15, the approximate time of eye opening in these animals, and was characterized by a sharp reduction in the rate of production of both ribbons and conventional synapses. During this phase ribbons achieved a final mean density of 113 ribbons/1,000 micrometer3 and conventionals achieved a final mean density of 250 synapses/1,000 micrometer3. Serial appeared in Phase II but remained at low densities.     

Synaptic analysis of inner plexiform layer in human retina

An ultrastructural synaptic analysis based on observations from 33 serial thin-sections of the inner plexiform layer in a volume sample of midperipheral human retina is presented. Of the 152 identifiable neuronal processes 7lpar;total 187) 14 were bipolar, 112 amacrine, and 26 ganglion. One hundred and forty-seven synapses were present — 31 bipolar (ribbon) and 116 amacrine (conventional). The ribbon synapses were divided on the basis of their postsynaptic processes into: amacrine, ganglion (16); amacrine, amacrine, ganglion (9) amacrine, amacrine (3) amacrine, amacrine, amacrine (1) ganglion (2), both incomplete in sections(. Of the 116 conventional synapses, 28% were amacrine-bipolar, 34% amacrine-amacrine and 38% were amacrine-ganglion. The occurrence of extra amacrine process(es) and the occasional absence of a ganglion cell dendrite at bipolar (ribbon) synapses, as well as the relative abundance of amacrine-amacrine (conventional) synapses, suggests a relatively greater role of amacrine cells in mediating visual transformations in the midperipheral human retina than has been described previously.     

GABA-ergic and glycinergic pathways in the inner plexiform layer of the goldfish retina

GABA-ergic and glycinergic circuitry in the inner plexiform layer of the goldfish retina was evaluated by electron microscopic autoradiography of 3H-GABA and 3H-glycine uptake, combined with retrograde horseradish peroxidase (HRP) labeling of ganglion cells. GABA-ergic and glycinergic synapses were found on labeled ganglion cells throughout the inner plexiform layer. This reinforces the idea that physiological evidence of GABA-ergic and glycinergic influence on a variety of ganglion cells in goldfish and carp often reflects direct inputs. Double-labeled synapses are presented as evidence of direct type Ab amacrine cell input to on-center ganglion cells. At least one population of type Aa sustained-off GABA-ergic amacrine cell is proposed, on the basis of profuse GABA-ergic inputs onto bipolar cells in sublamina a. Similar GABA-labeled profiles are shown to synapse onto HRP-labeled probable off-center ganglion cells. Thus GABA-ergic amacrine cells not only provide the predominant feedback to depolarizing (on-center) and hyperpolarizing (off-center) bipolar cells but also provide feed-forward inputs to on- and off-center ganglion cells. Large-caliber GABA-ergic dendrites present in both sublaminae a and b resemble those expected of a previously described bistratified, transient amacrine cell. These processes synapse onto HRP-labeled ganglion cell profiles in both sublaminae. Two morphologies of glycinergic amacrine cell are proposed on the basis of light microscopic autoradiography, 1) the previously described small pyriform cell and 2) a multipolar cell. The differential connectivity of the glycinergic neurons described, however, remains indistinguishable. Whereas abundant glycinergic inputs to ganglion cells occur throughout the inner plexiform layer, contacts between glycinergic profiles and bipolar cells are extremely rare. Therefore, interpreting the meaning of glycinergic input to ganglion cells will require further study of amacrine cell circuitry.     

Structure of the synaptic membranes in the inner plexiform layer of the retina: A freeze-fracture study in monkeys and rabbits

The internal structure of the synaptic membranes in the inner plexiform layer (IPL) of the retina of monkeys and rabbits was studied with the freeze-fracturing technique. In ribbon synapses, the presynaptic active zone is characterized by an aggregate of P-face particles, images of synaptic vesicle exocytosis, and forming coated vesicles which occupy distinct, contiguous membrane domains from apex to base of the synaptic ridge. The postsynaptic membrane contains a prominent aggregate of homogeneous particles which remain associated with the E-face. In the presynaptic membrane of conventional synapses, images of synaptic vesicle exocytosis are intermingled with large P-face particles, whereas forming coated vesicles surround the active zone. Three types of internal organization characterize the postsynaptic membrane of conventional synapses. Usually, the postsynaptic membrane exhibits the same internal structure as the surrounding nonjunctional plasmalemma. A second, less common type of conventional synapse contains a loose aggregate of heterogeneous particles which remain associated with the P-face. Finally, synapses were exceptionally found which are macular in shape and contain an aggregate of E-face particles within the postsynaptic membrane. The freeze-fracture evidence suggests that the axonal endings of bipolar cells—or at least some of them—make excitatory synapses, whereas the vast majority of amacrine cell dendrites make inhibitory synapses. Additional specializations of the cell surface in the IPL include gap junctions, puncta adhaerentia, subsurface cisterns, and cell corner aggregates.     

On the development of the stratification of the inner plexiform layer in the chick retina

This study investigated the development of the subdivision of the chick inner plexiform layer (IPL). The approach included an immunohistological analysis of the temporal and spatial expressions of choline acetyltransferase, of the neural-glial-related and neural-glial cell adhesion molecules (NrCAM and NgCAM, respectively) and axonin-1, and of inwardly rectifying potassium (Kir) channels in 5- to 19-day-old (E5-E19) embryos. Ultrastructural investigations evaluated whether synaptogenesis accompanies the onset of differentiation of the IPL. We found that the differentiation of the IPL started at E9. Distinct cholinergic strata appeared, NrCAM immunoreactivity showed a poorly defined stratification, and Kir3.2 was expressed in the IPL and in the inner nuclear layer. From E10 until late E14, NgCAM- and axonin-1-immunoreactive strata emerged in an alternating sequence from the outer to the inner IPL. During this period, the NrCAM pattern sharpened, and eventually five bands of weaker and stronger immunoreactivity were found. Conventional synapses formed at the beginning of E9, and stratification of the IPL also began on the same day at the same location. Synaptogenesis and stratification followed a gradient from the central to the peripheral retina. The topographic course of differentiation of the IPL generally corresponded to the course of maturation of ganglion and amacrine cells. Synaptogenesis and the expression of G-protein-gated Kir3.2 channels accompanied the onset of stratification. These events coincide with the occurrence of robust and rhythmic spontaneous neuronal activity. The subsequent differentiation of the IPL seemed to be orchestrated by several mechanisms.     

Synaptic connections of rod bipolar cells in the inner plexiform layer of the rabbit retina

We have reconstructed from electron micrographs of a continuous series of thin sections the synaptic connections of the axonal arborizations of all the rod bipolar cells contained in a small region of the retina of the rabbit. We observed that all rod bipolars share the same pattern of connectivity and are probably functionally equivalent. As a rule, they do not contact ganglion cells. Their prevalent synaptic output is on narrow-field, bistratified, and indoleamine-accumulating amacrine cells. Their dominant inputs are the reciprocal synapses from the indoleamine-accumulating amacrines, but they also receive a sizable number of synaptic contacts from other, non-reciprocal, amacrine cells. The lateral spread of scotopic signals at the synapse between rod bipolars and narrow-field, bistratified amacrines is small. Finally, in the rabbit, as in the cat, a narrow-field, bistratified amacrine is inserted in series along the rod pathway.     

Synaptic distribution of ionotropic glutamate receptors in the inner plexiform layer of the primate retina

The distribution and synaptic clustering of glutamate receptors (GluRs) were studied in the inner plexiform layer (IPL) of the macaque monkey retina by using subunit specific antisera. A punctate immunofluorescence pattern was observed in the IPL for all subunits tested, and electron microscopy confirmed that the immunoreactive puncta represent clustering of receptors at sites postsynaptic to the bipolar cell ribbon synapses (dyads). Usually only one of the two postsynaptic processes at the dyads expressed a given subunit. Immunoreactive GluR2, GluR2/3, and GluR4 puncta were found at high density throughout the IPL and are probably expressed at every dyad. The GluR1 subunit was expressed at lower density. The N-methyl-D-aspartate (NMDA) receptor subunits NR2A and NR1C2' were restricted to synapses localized in two broad bands in the center of the IPL. They were often colocalized with GluR2/3 and GluR4 subunits. The orphan receptor subunits delta 1/2 predominated in three horizontal bands. The kainate receptor subunits GluR6/7 were clustered in large postsynaptic densities adjacent to bipolar cell axon terminals but lacking a synaptic ribbon on the presynaptic side. This might represent a conventional synapse made by a bipolar axon terminal. The results suggest that GluR2/3 and GluR4, together with NMDA receptors, are preferentially expressed on ganglion cell dendrites, whereas kainate receptors and the delta 1/2 subunits are mostly localized on amacrine cell processes.     

Development of the rhesus monkey retina: II. A three-dimensional analysis of the sequences of synaptic combinations in the inner plexiform layer

The inner plexiform layer (IPL) of the retina provides a useful model for ultrastructural analysis of synaptic development. In primates, the IPL consists of numerous combinations of neuronal contacts that assume the morphological configuration of either conventional or ribbon synapses. We have determined the sequential development of these combinations by analyzing serial electron microscopic sections from fetal rhesus monkeys. Our analysis reveals an orderly emergence of various pre- and postsynaptic elements: (1) patches of dense filamentous membrane first appear on the dendrites of ganglion (G) cells; (2) membrane densities on ganglion cell dendrites then become apposed to amacrine (A) cell processes still lacking their own membrane densities and synaptic vesicles; (3) amacrine cell processes acquire membrane specializations associated with vesicles at the sites apposing ganglion cell dendrites, thereby establishing the first morphologically complete, A----G subtype of conventional synapse; (4) pairs of amacrine cell processes form A----A subtypes of conventional synapses; (5) next, monad ribbon synapses are established between bipolar (B) and ganglion or amacrine cell processes (B----G; B----A); (6) the three subclasses of dyad ribbon synapses (B----GG; B----GA; B----AA) are subsequently formed by the introduction of additional amacrine or ganglion cell processes in the dyad synapse; (7) concurrently, processes of some amacrine and interplexiform (I) cells form a feedback circuit with bipolar cell axons (A----B; I----B), thereby completing the synaptic microcircuitry of the IPL. Present findings provide evidence that the sequence of synaptic differentiation in the IPL proceeds from the postsynaptic to the presynaptic site. Furthermore, lateral interactions (A----G and A----A) are established prior to the formation of the "straight signal" pathway from photoreceptors (P) via bipolar cells to ganglion cells (P----B----G). Observed developmental events provide new insight into the order of establishment of local neuronal circuits in the primate retina.     

ON cone bipolar cell axonal synapses in the OFF inner plexiform layer of the rabbit retina

Analysis of the rabbit retinal connectome RC1 reveals that the division between the ON and the OFF inner plexiform layer (IPL) is not structurally absolute. ON cone bipolar cells make noncanonical axonal synapses onto specific targets and receive amacrine cell synapses in the nominal OFF layer, creating novel motifs, including inhibitory crossover networks. Automated transmission electron microscopic imaging, molecular tagging, tracing, and rendering of ～400 bipolar cells reveals axonal ribbons in 36% of ON cone bipolar cells, throughout the OFF IPL. The targets include γ‐aminobutyrate (GABA)‐positive amacrine cells (γACs), glycine‐positive amacrine cells (GACs), and ganglion cells. Most ON cone bipolar cell axonal contacts target GACs driven by OFF cone bipolar cells, forming new architectures for generating ON–OFF amacrine cells. Many of these ON–OFF GACs target ON cone bipolar cell axons, ON γACs, and/or ON–OFF ganglion cells, representing widespread mechanisms for OFF to ON crossover inhibition. Other targets include OFF γACs presynaptic to OFF bipolar cells, forming γAC‐mediated crossover motifs. ON cone bipolar cell axonal ribbons drive bistratified ON–OFF ganglion cells in the OFF layer and provide ON drive to polarity‐appropriate targets such as bistratified diving ganglion cells (bsdGCs). The targeting precision of ON cone bipolar cell axonal synapses shows that this drive incidence is necessarily a joint distribution of cone bipolar cell axonal frequency and target cell trajectories through a given volume of the OFF layer. Such joint distribution sampling is likely common when targets are sparser than sources and when sources are coupled, as are ON cone bipolar cells. J. Comp. Neurol. 521:977–1000, 2013. © 2012 Wiley Periodicals, Inc.     

The inner plexiform layer of the vertebrate retina: a quantitative and comparative electron microscopic analysis

The inner plexiform layer of human, monkey, cat, rat, rabbit, ground squirrel, frog and pigeon retinas was studied by electron microscopy. All showed the same qualitative synaptic arrangements: bipolar cells made dyad ribbon synapses onto amacrine and ganglion cells; amacrine cells made conventional synaptic contacts onto bipolar, ganglion cells; amacrine cells montage of electron micrographs through the full thickness of the inner plexiform layer were made for each species and were scored for synaptic contacts. Both absolute and relative quantitative differences were found between species. The ratio of amacrine cell (conventional) synapses to bipolar cell (ribbon) synapses, the absolute number of amacrine cell synapses and the number of inter-amacrine cell synapses were all found to be higher in those animals which are known to have relatively complex retinal ganglion cell receptive field properties. It is suggested that the amacrine cell is involved in mediating complex visual transformations in certain vertebrate retinas.     

Glutamate receptors at bipolar synapses in the inner plexiform layer of primate retina: light microscopic analysis

At least 10 different types of bipolar cells have been distinguished in the primate retina. The axon terminals of these cells stratify in distinct strata in the inner plexiform layer and are involved in parallel pathways to distinct types of ganglion cells. Ionotropic glutamate receptor (GluR) subunits also show a stratified distribution in the inner plexiform layer. Here, we investigated whether different types of bipolar cells are associated with different types of ionotropic glutamate receptors in the inner retina of a New World primate, the common marmoset Callithrix jacchus. Vertical cryostat sections through central retina were double labeled with immunohistochemical markers for bipolar cell types and with antibodies to alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunits GluR1 to 4, kainate receptor subunits GluR6/7, and the NR1C2' subunit of the N-methyl-D-aspartate (NMDA) receptor. The axon terminals of bipolar cell types were reconstructed from confocal sections, and the colocalized immunoreactive puncta were quantified. For all bipolar cell types, immunoreactive puncta for the AMPA receptor subunits GluR2, 2/3, and 4 were colocalized at highest densities, whereas GluR1-immunoreactive puncta were expressed at very low densities. The kainate receptor subunits GluR6/7 were predominantly associated with diffuse bipolar (DB6) and rod bipolar cells. The NMDA receptor subunit NR1C2' was specifically colocalized with flat midget and DB3 axons. These findings suggest that rod and cone bipolar cell types contribute to multiple but distinct glutamate receptor pathways in primate retina.     

Synaptic connectivity of the diffuse bipolar cell type DB6 in the inner plexiform layer of primate retina

Diffuse bipolar cells in primate retina receive synaptic input from multiple cones and provide output to ganglion cells. Diffuse bipolar cells can be subdivided into six types (DB1-DB6) according to the stratification of their axon terminals in the inner plexiform layer, but their synaptic connectivity in the inner plexiform layer is not well understood. Here the stratification and synaptic connectivity of DB6 axon terminals were studied in the retinae of New World (marmoset) and Old World (macaque) monkeys. Immunohistochemical markers were applied to retinal sections. The sections were analyzed by confocal and deconvolution light microscopy as well as electron microscopy. The DB6 cells were identified with antibodies against CD15; rod bipolar cells were identified with antibodies against protein kinase Calpha (PKCalpha); and AII amacrine cells were identified with antibodies against calretinin. The axons of DB6 and rod bipolar cells occupy distinct regions in stratum 5 of the inner plexiform layer. The distal processes of calretinin-labeled AII cells are usually closely associated with rod bipolar axons but sometimes also with DB6 axons. Pre-embedding immunoelectron microscopy showed that the vast majority (over 86%) of the synaptic output of DB6 cells is onto amacrine cell processes, whereas less than 14% goes to ganglion cell processes. In double-labeled preparations DB6 axons occasionally made output onto calretinin-labeled amacrine processes. Thus it is possible that AII cells receive some input from DB6 cells.     

Development of the rhesus monkey retina. I. Emergence of the inner plexiform layer and its synapses

The emergence and differentiation of the inner plexiform layer (IPL) and the establishment of its synapses were analyzed in retinae of 18 rhesus monkeys ranging in age from the 55th embryonic day (E55) to 10 years. The IPL becomes recognizable by E65 as a thin acellular zone consisting of immature neurites and growth cones scattered within large extracellular spaces. In each specimen, apposing paired membrane specializations were classified as junctions without synaptic vesicles, conventional synapses, ribbon synapses, or gap junctions. Initially, at E65, the IPL consists of variety of immature cell processes that are interconnected exclusively by junctions without synaptic vesicles, at a density of 4.7/100 microns2. By E73, the IPL becomes more distinct and wider and contains 7.8 such junctions/100 microns2. Conventional synapses develop by the addition of vesicles to initially vesicle-free junctions. The first conventional synapses appear at E78. They increase in density from 1.5 to 3.2/100 microns2 between E78 and E84 and reach a density of 7.9/100 microns2 by E99. A rapid burst in synaptogenesis occurs in the IPL between E99 and E114; a density of 16.5/100 microns2 is reached, mainly due to accretion of conventional synapses. Ribbon synapses first become recognizable at E99, almost 3 weeks after the emergence of conventional synapses. By E114 they account for about 7% of all synaptic contacts in the IPL. The rate of synaptogenesis slows down during the last quarter of gestation; the adult level of about 24 contacts/100 microns2 is reached between E130 and E149. Of these, 72.2% are of conventional type, 15.4% are ribbon synapses, and 12.4% are junctions without vesicles. However, in the adult the density of junction without vesicles is only about one-half that found at E149. Gap junctions are absent during the initial and rapid phases of synaptogenesis; they appear abruptly, between E130 and E149, only after the density of chemical synapses in the IPL has reached the adult level. In the rhesus monkey, synaptogenesis begins several weeks later in the IPL than in its primary targets--the dorsal lateral geniculate nucleus and the superior colliculus (Hendrickson and Rakic, '77; Cooper and Rakic, '83). However, the rapid increase in density of conventional synapses in the IPL coincides with the segregation of retinal projections from right and left eyes in the geniculate nucleus (Rakic, '76) and with the elimination of the large surplus of retinal ganglion cell axons (Rakic and Reley, '83).     

Selective synaptic distribution of AMPA and kainate receptor subunits in the outer plexiform layer of the carp retina.

The subunit composition of ionotropic glutamate receptors (GluRs) is extremely diverse and responsible for the diversity of postsynaptic responses to the release of glutamate, which is the major excitatory neurotransmitter in the retina. To understand the functional consequences of this diversity, it is necessary to reveal the synaptic localization and subunit composition of GluRs. We have used immuno light and electron microscopy to localize AMPA and kainate (GluR1, GluR2/3, GluR4, GluR5-7) subunits in identified carp retinal neurons contributing to the outer plexiform layer. GluR1 could not be detected within the outer plexiform layer. Rod and cone horizontal cells all express only GluR2/3 at the tips of their invaginating dendrites. These receptors are also inserted into the membrane of spinules, light-dependent protrusions of the horizontal cell dendrites, flanking the synaptic ribbon of the cone synapse. Bipolar cells express GluR2/3, GluR4, and GluR5-7 at their terminal dendrites invaginating cone pedicles and rod spherules. Colocalization data suggest that each subunit is expressed by a distinct bipolar cell type. The majority of bipolar cells expressing these receptors seem to be of the functional OFF-type; however, in a few instances, GluR2/3 could also be detected on dendrites of bipolar cells that, based on their localization within the cone synaptic complex, appeared to be of the functional ON-type. The spatial arrangement of the different subunits within the cavity of the cone pedicle appeared not to be random: GluR2/3 was found predominantly at the apex of the cavity, GluR4 at its base and GluR5-7 dispersed between the two.     

Bar synapses and gap junctions in the inner plexiform layer: synaptic relationships of the interstitial amacrine cell of the retina of the cichlid fish, Astronotus ocellatus

The retina of the cichlid fish, Astronotus ocellatus, contains an unusual class of amacrine cell, the interstitial amacrine cell, which has its soma and processes restricted to a sublamina of the proximal inner plexiform layer. The interstitial amacrine cell is unique in making synapses which contain a presynaptic dense bar specialization. The interstitial amacrine cell makes reciprocal synapses with bipolar cell terminals and is presynaptic to other amacrine cells and to ganglion cell dendrites. Processes of interstitial amacrine cells are connected to each other by large gap junctions.