Photoreceptor Ribbon Synapses Research Articles

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.

Quantal mEPSCs and residual glutamate: how horizontal cell responses are shaped at the photoreceptor ribbon synapse

At the photoreceptor ribbon synapse, glutamate released from vesicles at different positions along the ribbon reaches the same postsynaptic receptors. Thus, vesicles may not exert entirely independent effects. We examined whether responses of salamander retinal horizontal cells evoked by light or direct depolarization during paired recordings could be predicted by summation of individual miniature excitatory postsynaptic currents (mEPSCs). For EPSCs evoked by depolarization of rods or cones, linear convolution of mEPSCs with photoreceptor release functions predicted EPSC waveforms and changes caused by inhibiting glutamate receptor desensitization. A low-affinity glutamate antagonist, kynurenic acid (KynA), preferentially reduced later components of rod-driven EPSCs, suggesting lower levels of glutamate are present during the later sustained component of the EPSC. A glutamate-scavenging enzyme, glutamic-pyruvic transaminase, did not inhibit mEPSCs or the initial component of rod-driven EPSCs, but reduced later components of the EPSC. Inhibiting glutamate uptake with a low concentration of DL-threo-beta-benzoyloxyaspartate (TBOA) also did not alter mEPSCs or the initial component of rod-driven EPSCs, but enhanced later components of the EPSC. Low concentrations of TBOA and KynA did not affect the kinetics of fast cone-driven EPSCs. Under both rod- and cone-dominated conditions, light-evoked currents (LECs) were enhanced considerably by TBOA. LECs were more strongly inhibited than EPSCs by KynA, suggesting the presence of lower glutamate levels. Collectively, these results indicate that the initial EPSC component can be largely predicted from a linear sum of individual mEPSCs, but with sustained release, residual amounts of glutamate from multiple vesicles pool together, influencing LECs and later components of EPSCs.

Temperature dependence of Ca v1.4 calcium channel gating

The CACNA1F gene encodes the pore-forming subunit of the L-type Ca v1.4 voltage-gated calcium channel (VGCC) and plays a central role in tonic vesicular release at photoreceptor ribbon synapses. The main objective of this study was to examine the effects of temperature on human Ca v1.4 cDNA clone VGCCs. With 20 mM Ba 2+ as charge carrier, increasing the temperature from 23 °C to 37 °C increases whole-cell conductance, shifts the voltage-dependence of activation to more hyperpolarized voltages, and accelerates the degree of recovery from inactivation over a given time, but does not significantly alter the half-inactivation potential (V h). The window current for Ca v1.4 was also shifted to more hyperpolarized voltages, observable from ∼−35 mV to +20 mV at 37 °C in 20 mM Ba 2+. Several comparable results were observed when characterizing Ca v1.2 at temperatures ranging from 23 °C to 37 °C. However, one difference between Ca v1.4 and Ca v1.2 was the temperature dependence of voltage-dependent inactivation kinetics. Increasing temperature from 23 °C to 37 °C accelerates Ca v1.4 inactivation kinetics ≈50-fold, whereas Ca v1.2 only accelerates ≈10-fold over the same temperature range. The time constant of inactivation (τ h) temperature coefficient (Q 10) was 18.8 for Ca v1.4 over a temperature range of 23° to 33 °C (corresponding to an activation energy E a=221 kJ/mol), compared with Ca v1.2 with a Q 10 of 3 (E a=90 kJ/mol) recorded under identical conditions. In addition, Ca v1.4 was also tested using 2 mM Ca 2+ as a charge carrier and similar changes in current–voltage Boltzmann parameters and gating kinetics were observed. Hence, despite the accelerated inactivation kinetics of Ca v1.4 channels observed at near physiological temperatures the window current is preserved and could allow for tonic glutamate release from photoreceptors in the retina during dark adapted conditions.

Mechanisms Contributing to Tonic Release at the Cone Photoreceptor Ribbon Synapse

Time-resolved capacitance measurements in combination with fluorescence measurements of internal calcium suggested three kinetic components of release in acutely isolated cone photoreceptors of the tiger salamander. A 45-fF releasable pool, corresponding to about 1,000 vesicles, was identified. This pool could be depleted with a time constant of a few hundred milliseconds and its recovery from depletion was quite rapid (tau approximately 1 s). The fusion of vesicles in this pool was blocked by low-millimolar EGTA. Endocytosis was sufficiently slow that it is likely that refilling of the releasable pool occurred from preformed vesicles. A second, slower component of release (tau(depletion) approximately 3 s) was identified that was approximately twice the size of the releasable pool. This pool may serve as a first reserve pool that replenishes the releasable pool. Computer simulations indicate that the properties of the releasable and first reserve pools are sufficient to maintain synaptic signaling for several seconds in the face of near-maximal stimulations and in the absence of other sources of vesicles. Along with lower rates of depletion, additional mechanisms, such as replenishment from distal reserve pools and the fast recycling of vesicles, may further contribute to the maintenance of graded, tonic release from cone photoreceptors.

Structural and functional remodeling in the retina of a mouse with a photoreceptor synaptopathy: plasticity in the rod and degeneration in the cone system

Knowledge about the plastic and regenerative capacity of the retina is of key importance for therapeutic approaches to restore vision in patients who suffer from degenerative retinal diseases. In the retinae of mice, mutant for the presynaptic scaffolding protein Bassoon, signal transfer at photoreceptor ribbon synapses is disturbed due to impaired ribbon attachment to the active zone. In a long-term study we observed, with light and electron microscopic immunocytochemistry and electroretinographic recordings, two overlapping events in the Bassoon mutant retina, i.e. loss of photoreceptor synapses in the outer plexiform layer, and structural remodeling and formation of ectopic photoreceptor synapses in the outer nuclear layer, a region usually devoid of synapses. Formation of ectopic synaptic sites starts around the time when photoreceptor synaptogenesis is completed in wild-type mice and progresses throughout life. The result is a dense plexus of ectopic photoreceptor synapses with significantly altered but considerable synaptic transmission. Ectopic synapse formation is led by the sprouting of horizontal cells followed by the extension of rod bipolar cell neurites that fasciculate with and grow along the horizontal cell processes. Although only the rod photoreceptors and their postsynaptic partners show structural and functional remodeling, our study demonstrates the potential of the retina for long-lasting plastic changes.

Cloning and functional analysis of Pikachurin: A novel Agrin/Perlecan-related protein localized at the active zone of retinal photoreceptor ribbon synapse

Cloning and functional analysis of Pikachurin: A novel Agrin/Perlecan-related protein localized at the active zone of retinal photoreceptor ribbon synapse

Rod bipolar cells and horizontal cells form displaced synaptic contacts with rods in the outer nuclear layer of the nob2 retina.

The nob2 mouse carries a null mutation in the Cacna1f gene, which encodes the pore-forming subunit of the L-type calcium channel, Ca(v)1.4. The loss of the electroretinogram b-wave in these mice suggests a severe reduction in transmission between photoreceptors and second-order neurons in the retina and supports a central role for the Ca(v)1.4 calcium channel at photoreceptor ribbon synapses, to which it has been localized. Here we show that the loss of Ca(v)1.4 leads to the aberrant outgrowth of rod bipolar cell dendrites and horizontal cell processes into the outer nuclear layer (ONL) of the nob2 retina and to the formation of ectopic synaptic contacts with rod photoreceptors in the ONL. Ectopic contacts are predominantly between rods and rod bipolar cells, with horizontal cell processes also present at some sites. Ectopic contacts contain apposed pre- and postsynaptic specializations, albeit with malformed synaptic ribbons. Cone photoreceptor terminals do not participate in ectopic contacts in the ONL. During retinal development, ectopic contacts appear in the days after eye opening, appearing progressively farther into the ONL at later postnatal stages. Ectopic contacts develop at the tips of rod bipolar cell dendrites and are less frequently associated with the tips of horizontal cell processes, consistent with the adult phenotype. The relative occurrence of pre- and postsynaptic markers in the ONL during development suggests a mechanism for the formation of ectopic synaptic contacts that is driven by the retraction of rod photoreceptor terminals and neurite outgrowth by rod bipolar cell dendrites.

Mutation in the Auxiliary Calcium-Channel Subunit CACNA2D4 Causes Autosomal Recessive Cone Dystrophy

Retinal signal transmission depends on the activity of high voltage-gated l-type calcium channels in photoreceptor ribbon synapses. We recently identified a truncating frameshift mutation in the Cacna2d4 gene in a spontaneous mouse mutant with profound loss of retinal signaling and an abnormal morphology of ribbon synapses in rods and cones. The Cacna2d4 gene encodes an l-type calcium-channel auxiliary subunit of the alpha (2) delta type. Mutations in its human orthologue, CACNA2D4, were not yet known to be associated with a disease. We performed mutation analyses of 34 patients who received an initial diagnosis of night blindness, and, in two affected siblings, we detected a homozygous nucleotide substitution (c.2406C-->A) in CACNA2D4. The mutation introduces a premature stop codon that truncates one-third of the corresponding open reading frame. Both patients share symptoms of slowly progressing cone dystrophy. These findings represent the first report of a mutation in the human CACNA2D4 gene and define a novel gene defect that causes autosomal recessive cone dystrophy.

A Highly Ca 2+-Sensitive Pool of Vesicles Contributes to Linearity at the Rod Photoreceptor Ribbon Synapse

Studies of the properties of synaptic transmission have been carried out at only a few synapses. We analyzed exocytosis from rod photoreceptors with a combination of physiological and ultrastructural techniques. As at other ribbon synapses, we found that rods exhibited rapid kinetics of release, and the number of vesicles in the releasable pool is comparable to the number of vesicles tethered at ribbon-style active zones. However, unlike other previously studied neurons, we identified a highly Ca 2+-sensitive pool of releasable vesicles with a relatively shallow relationship between the rate of exocytosis and [Ca 2+] i that is nearly linear over a presumed physiological range of intraterminal [Ca 2+]. The low-order [Ca 2+] dependence of release promotes a linear relationship between Ca 2+ entry and exocytosis that permits rods to relay information about small changes in illumination with high fidelity at the first synapse in vision.

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 neuronal calcium sensor-1 in the developing chick retina.

Neuronal calcium sensor-1 (NCS-1) is a Ca(2+) binding protein that has been implicated in the regulation of neurotransmission and synaptogenesis. In this study we investigated the developmental expression and localization of NCS-1 in the chick retina. Single- and double-labeling experiments with three-dimensional reconstruction as well as ultrastructural data of the distribution of NCS-1 suggest that this protein is also involved in axonal process outgrowth. We found an early expression of NCS-1 in ganglion cells and their axons, in amacrine, and in horizontal cells, whereas photoreceptors were immunonegative at embryonic stages. In the early posthatching days we found strong immunostaining for NCS-1 in horizontal cells and their processes in the outer plexiform layer. In contrast, synaptic vesicle protein 2 (SV2) was prominent only in photoreceptor synaptic terminals. Ultrastructural analysis confirmed that NCS-1 was localized postsynaptically in horizontal cell processes, whereas presynaptic terminals were immunonegative. However, at late posthatching days we observed that photoreceptor ribbon synapses (from rods and/or cones) also expressed NCS-1. Thus the results support the notion that NCS-1 is involved in neuronal process outgrowth and is localized in pre- and postsynaptic compartments including mature photoreceptor synapses.

L‐type calcium channels in the photoreceptor ribbon synapse: Localization and role in plasticity

L‐type calcium channels in the photoreceptor ribbon synapse: Localization and role in plasticity

L-type calcium channels in the photoreceptor ribbon synapse: Localization and role in plasticity

Calcium (Ca(2+)) influx through voltage-gated Ca(2+)channels stimulates a variety of neural activities, including process outgrowth, neurotransmission, and synaptic plasticity. In general, L-type channels control Ca(2+) influx into the soma and dendrites, whereas other Ca(2+) channel types control presynaptic activities. Neurons that make ribbon synapses, however, are among a select group of nerve cells whose presynaptic Ca(2+)-dependent secretion is linked to L-type channels. Recently, photoreceptor ribbon synapses have been shown to be capable of dramatic structural remodeling and neuritic outgrowth. Here, we have examined 1) the distribution of dihydropyridine (DHP)-sensitive (L-type) Ca(2+) channels in photoreceptor presynaptic structures and 2) the role of these channels in axonal plasticity and process outgrowth in culture. Using anti-alpha(1C) and the fluorescent dihydropyridine, (-)-DM-BODIPY DHP, L-type channels were localized in the outer plexiform layer of retinal sections and in presynaptic terminals of freshly isolated photoreceptors. In the rod terminal, dense patches of label were present; their distribution and number matched that of synaptic ribbons. After 1-7 days in vitro, punctate alpha(1C) staining occurred along newly formed neurites and presynaptic varicosities. Functional channels were present throughout the culture period, as determined by fura-2 imaging. Channel blockage by nicardipine, a DHP antagonist, inhibited axonal remodeling. Specifically, it prevented axon retraction and lamellipodium formation, reduced neurite growth, and produced long, thin processes on some, primarily cone, photoreceptors. L-type Ca(2+) channel activity, therefore, not only stimulates neurotransmission but contributes to presynaptic structural plasticity at the ribbon synapse.

Gamma-atrial natriuretic peptide 1-25 is found in bipolar cells in turtle and rat retinas.

Immunocytochemistry was used to reveal a population of bipolar cells that contain gamma-atrial natriuretic peptide 1-25 (gamma-ANP) in turtle retina. This same antibody was also used in rat retina as a comparative control. The retinas were examined by both conventional light microscopy and confocal microscopy with double-labeling to determine whether protein kinase C-alpha-like immunoreactivity (PKC-alpha-LI) was colocalized with the gamma-ANP-LI. Some thick sections of turtle retina immunostained with only the gamma-ANP antibody were also examined by electron microscopy. In rat, a subpopulation of bipolar cells with axons terminating close to the ganglion cell layer was labeled. Double-labeling experiments indicated that the gamma-ANP-LI and PKC-alpha-LI were colocalized in rat retina, and thus all the bipolar cells with gamma-ANP-LI were rod bipolar cells. In turtle, the gamma-ANP antibody labeled certain bipolar cells that were characterized by bistratified axon terminals arborizing on the borders of strata S2/3 and S3/4 in the inner plexiform layer (IPL). Double labeling with PKC-alpha antibody indicated that bipolar cells with gamma-ANP-LI were not the same bipolar cell types with PKC-alpha-LI. Thus, gamma-ANP-LI appears to be a new marker for a distinct type of bipolar cell in turtle retina. At the ultrastructural level, the gamma-ANP-LI was visible throughout the cytoplasm of the bipolar cells from dendrites to axon terminals. In the outer plexiform layer (OPL), labeled dendrites contacted photoreceptor pedicles almost exclusively at narrow-cleft basal junctions, but infrequently formed the central element at a photoreceptor ribbon synapse. In the IPL, axon terminals with gamma-ANP-LI made ribbon synapses onto a combination of amacrine and ganglion cells. Since narrow-cleft basal junctions and photoreceptor ribbon-related junctions are known to be associated with ON-center bipolar cells in turtle, and since the axon terminals of bipolars with gamma-ANP-LI stratify primarily in the ON-strata of the IPL, we suggest that these cells are likely to be ON-center cells. It is possible that the gamma-ANP may be involved in regulating the activity of Na+/K+ ATPase or in the modulation of cGMP levels.

The Kinesin Motor KIF3A Is a Component of the Presynaptic Ribbon in Vertebrate Photoreceptors

Kinesin motors are presumed to transport various membrane compartments within neurons, but their specific in vivo functions, cargoes, and expression patterns in the brain are unclear. We have investigated the distribution of KIF3A, a member of the heteromeric family of kinesins, in the vertebrate retina. We find KIF3A at two distinct sites within photoreceptors: at the basal body of the connecting cilium axoneme and at the synaptic ribbon. Immunoelectron microscopy of the photoreceptor ribbon synapse shows KIF3A to be concentrated both at the ribbon matrix and on vesicles docked at the ribbon, a result that is consistent with the presence of both detergent-extractable and resistant KIF3A fractions at these synapses. KIF3A is also present in the inner plexiform layer, again at presynaptic ribbons. These findings suggest that within a single cell, the photoreceptor, one kinesin polypeptide, KIF3A, can serve two distinct functions, one specific for ribbon synapses.

Immunocytochemical localization of the synapse-associated protein SAP102 in the rat retina

An elaborate network of transmitter receptors, synapse associated proteins (SAPs), and cytoskeletal elements, generally known as the postsynaptic density, is involved with efficient synaptic signaling. The localization of the synapse associated protein SAP102 was studied in the rat retina by using immunocytochemical methods. Immunofluorescence for SAP102 was most prominent in the inner plexiform layer (IPL). It had a punctate appearance, suggesting a synaptic clustering of SAP102 in the IPL. Electron microscopy by use of pre-embedding immunocytochemistry showed that SAP102 is concentrated in the IPL in processes which are postsynaptic at bipolar cell ribbon synapses (dyads). As a rule, only one of the two postsynaptic members of the dyad was labeled for SAP102. Double-labeling experiments were performed in order to find out whether SAP102 is involved with the clustering the N-methyl-D-aspartate (NMDA) receptor 2A subunit (NR2A). Only a fraction (approximately 23%) of the SAP102 clusters expressed NR2A, suggesting SAP102 is also associated with other subunits or receptors. Distinct SAP102 labeling was also present in horizontal cell processes in the outer plexiform layer (OPL), which are inserted as lateral elements into photoreceptor ribbon synapses (triads). The optic nerve fibre layer was also diffusely immunoreactive for SAP102. The postsynaptic aggregation of SAP102 at bipolar cell dyads and at photoreceptor triads suggests SAP102 is associated with the clustering of transmitter receptors. However, the labeling of the optic nerve fibre layer indicates additional functions of SAP102 in the retina.

Immunocytochemical localization of the synapse‐associated protein SAP102 in the rat retina

An elaborate network of transmitter receptors, synapse associated proteins (SAPs), and cytoskeletal elements, generally known as the postsynaptic density, is involved with efficient synaptic signaling. The localization of the synapse associated protein SAP102 was studied in the rat retina by using immunocytochemical methods. Immunofluorescence for SAP102 was most prominent in the inner plexiform layer (IPL). It had a punctate appearance, suggesting a synaptic clustering of SAP102 in the IPL. Electron microscopy by use of pre-embedding immunocytochemistry showed that SAP102 is concentrated in the IPL in processes which are postsynaptic at bipolar cell ribbon synapses (dyads). As a rule, only one of the two postsynaptic members of the dyad was labeled for SAP102. Double-labeling experiments were performed in order to find out whether SAP102 is involved with the clustering the N-methyl-D-aspartate (NMDA) receptor 2A subunit (NR2A). Only a fraction (approximately 23%) of the SAP102 clusters expressed NR2A, suggesting SAP102 is also associated with other subunits or receptors. Distinct SAP102 labeling was also present in horizontal cell processes in the outer plexiform layer (OPL), which are inserted as lateral elements into photoreceptor ribbon synapses (triads). The optic nerve fibre layer was also diffusely immunoreactive for SAP102. The postsynaptic aggregation of SAP102 at bipolar cell dyads and at photoreceptor triads suggests SAP102 is associated with the clustering of transmitter receptors. However, the labeling of the optic nerve fibre layer indicates additional functions of SAP102 in the retina. J. Comp. Neurol. 397:326–336, 1998. © 1998 Wiley-Liss, Inc.

Localization of nNOS in photoreceptor, bipolar and horizontal cells in turtle and rat retinas.

Neuronal nitric oxide synthase (nNOS), an enzyme that synthesizes NO, has been found in the outer retina using light microscopic immunocytochemistry, but its subcellular localization is unknown. We used electron immunocytochemistry to examine nNOS-like immunoreactivity (nNOS-LI) in the outer plexiform layer of turtle and rat retinas. In turtle, nNOS-LI was present in some bipolar and horizontal cell processes at photoreceptor ribbon synapses and at basal junctions between photoreceptors. In rat, nNOS-LI was present in some rod bipolar and B-type horizontal cell axon terminals at rod ribbon synapses. These results indicate that in vertebrates, NO is produced by all of the major nerve cell types in the outer retina at specific synaptic contacts.

Distribution of synaptic markers in the retina: Implications for synaptic vesicle traffic in ribbon synapses

Synapses in the retina are present in two layers that can easily be distinguished morphologically: the thin outer plexiform layer (OPL) containing the photoreceptor ribbon synapses, and the more complex inner plexiform layer (IPL) containing mostly conventional synapses. In the current study we have used the exquisite spatial separation between ribbon and conventional synapses in the retina to investigate by immunocytochemistry the protein components of these two types of synapses. Our results show that all of the synaptic vesicle proteins tested are present in the ribbon synapses of photoreceptors except for the synapsins which were previously described to be absent from ribbon synapses. Thus, synaptic vesicles of ribbon and conventional synapses are likely to be very similar molecularly in spite of their different modes of trafficking. Furthermore, proteins with a likely function in synaptic vesicle docking and fusion (SNAP-25, munc-18) and endocytosis (dynamin) were also highly enriched in ribbon synapses, indicating that the basic machineries for synaptic membrane traffic are similar in ribbon and conventional synapses. Interestingly, photoreceptor cells contained only low concentrations of syntaxin I (which functions in the synaptic vesicle fusion complex) and GDI (which regulates the membrane association of rab-proteins as a function of GTP/GDP binding). These results raise the possibility that photoreceptors express other isoforms of these proteins, or that the functions of these proteins are not required for the tonical release mode of their synapses. Finally, Ca 2+/calmodulin-dependent kinase II was highly enriched in the photoreceptor synapses in spite of the absence of synapsin I, one of its major neuronal substrates, suggesting that the major synaptic function of this kinase is not related to synapsin. Together our results indicate that ribbon and conventional synapses utilize largely parallel mechanisms of membrane traffic.

Synaptic microcircuitry of bipolar and amacrine cells with serotonin-like immunoreactivity in the retina of the turtle, Pseudemys scripta elegans.

Although serotonin is thought to be a neurotransmitter in a number of retinal systems, much of the precise synaptic connectivity of serotonergic neurons is unknown. To address this issue, we used an antiserum directed against serotonin to label serotonergic bipolar and amacrine cells in the turtle retina. Light-microscopic analysis of labeled amacrine and bipolar cells indicated that both had bistratified dendritic arborizations primarily in stratum 1 and in strata 4/5 of the inner plexiform layer. Ultrastructural analysis of the neurocircuitry of these cells indicated that the processes of labeled bipolar cells in the outer plexiform layer made basal junction contacts with photoreceptor terminals. Only in rare instances did labeled bipolar cells processes invaginate near photoreceptor ribbon synapses. Processes of labeled bipolar cells received both conventional and small ribbon synaptic contacts in the outer plexiform layer. Bipolar cell processes in stratum 1 of the inner plexiform layer synapsed onto either amacrine/amacrine or amacrine/ganglion cell dyads, and made rare ribbon synaptic contacts onto labeled amacrine cell processes. Synaptic inputs to serotonergic bipolar cells in stratum 1 were from unlabeled bipolar and amacrine cells. Bipolar cell contacts in strata 4/5 were similar to those in stratum 1, but were fewer in number and no bipolar cell inputs were seen. Labeled amacrine cell output in both strata was onto other unlabeled amacrine cells and ganglion cells; but synaptic outputs to unlabeled bipolar cells were only seen in strata 4/5. In both strata 1 and 4/5, synaptic inputs to labeled amacrine cells were from both unlabeled amacrine cells and labeled bipolar cells. The serotonergic amacrine cells had many more synaptic interactions in stratum 1 than in strata 4/5 which supports the role of serotonergic bipolar cells in the OFF pathway of retinal processing. Interactions between serotonergic bipolar and amacrine cells may play an important role in visual processing.