Retinal Circuitry Research Articles

Morphological characterization of the retina of the CNGA3(-/-)Rho(-/-) mutant mouse lacking functional cones and rods.

To assess the structural changes in the retina caused by a functional blockade of rods and cones and to document the time course of their degeneration. Double knockout mice were generated by cross-breeding CNGA3(-/-) mice with Rho(-/-) mice. Retinas of mutant and wild-type mice from 3 weeks up to 12 months of age were studied by confocal light and electron microscopy. The retinas were immunostained with cell-type-specific markers and with antibodies against synapse-associated proteins and transmitter receptors. In 3-week-old CNGA3(-/-)Rho(-/-) mice, retinal layers showed normal structural organization, and photoreceptors established normal synaptic contacts. Until postnatal week (Pw)7, presynaptic markers and postsynaptic glutamate receptors were well expressed at the photoreceptor terminals. Photoreceptor degeneration started at Pw4, progressing to an almost complete loss by 3 months. Rod spherules showed an increase in the number of synaptic ribbons and postsynaptic elements during this early stage of degeneration, and horizontal cell processes grew into the outer nuclear layer. At later stages of retinal degeneration, the inner plexiform layer (IPL) was also affected. Rod bipolar cell axon terminals showed morphologic alterations, but the stratification pattern of cone bipolar cell axons and amacrine cell processes appeared unaffected. Transmitter receptors (GlyRalpha3, GABA(A) alpha2, GluR2/3) showed no obvious changes in the distribution and density of their synaptic clusters throughout the IPL at postnatal month 12. The normal structural and synaptic organization of the mutant retina at Pw3 suggests that photoreceptor light responses are not essential for the development of the retinal circuitry. However, functional photoreceptors are necessary for the maintenance of rods and cones and their contacts in the OPL, because they degenerate almost completely by 3 months after birth. Degenerative changes can also be observed in the IPL; however, they appear to have a slower time course and by 12 months of age the IPL circuitry appears to be surprisingly intact.

Neurokinin 1 receptor expression and substance p physiological actions are developmentally regulated in the rabbit retina

We investigated the expression of the substance P (SP) receptor (the neurokinin 1 receptor, NK1 receptor) and SP functional effects in developing rabbit retinas. NK1 receptors in adult retinas were in a population of cone bipolar cells and in dopaminergic amacrine cells, as previously described. In contrast, at birth and at postnatal day (PND) 6, NK1 receptors were exclusively expressed by cholinergic amacrine and displaced amacrine cells. NK1 receptor expression in cholinergic cells was still observed at PND10 (eye opening), while at PND21 it was confined to cholinergic cells of the inner nuclear layer. Starting at PND10, NK1 receptors were also in bipolar cells and in dopaminergic amacrine cells. A fully mature NK1 receptor expression pattern was observed at PND35. Dopamine release was assessed in isolated retinas in the presence of SP, the NK1 receptor agonist GR73632 or the NK1 receptor antagonist GR82334. At PND35, extracellular dopamine was significantly increased by 10 μM SP or 0.01–100 μM GR73632, and it was decreased by 0.01–10 μM GR82334. No effects were detected in developing retinas up to PND21. Ca 2+ imaging experiments were performed in single cholinergic cells identified by their “starburst” morphology in perinatal retinas. Intracellular Ca 2+ levels were significantly increased by 1 μM SP or GR73632. This effect was reversibly inhibited by 1 μM GR82334. These data demonstrate that both NK1 receptor expression and SP physiological actions are developmentally regulated in the retina. SP neurotransmission in the immature retina may subserve developmental events, and SP is likely to represent an important developmental factor for the maturation of retinal neurons and circuitries.

Light deprivation suppresses the light response of inner retina in both young and adult mouse.

The retinal synaptic network continues its development after birth in mammals. Recent studies show that postnatal development of retinal circuitry depends on visual stimulation. We sought to determine whether there is a time period during which the retina shows evidence of increased plasticity. We examined the effects of light deprivation on the retinal light response of mouse retina using electroretinogram (ERG) measurements. Our results showed that dark rearing mice from birth to postnatal day (P) 30, 60, and 90 suppressed the amplitudes of oscillatory potentials (OPs) and the magnitudes of suppression were age independent. In addition, dark-rearing-produced suppression of OP amplitudes can be completely reversed in both young and adult mice by returning them to cyclic light/dark conditions for 1 to 2 weeks. However, the recovery time course was age dependent with younger animals needing a longer time to achieve a full recovery. Furthermore, dark rearing of P60 mice raised under cyclic light/dark conditions for 30 days resulted in a similar magnitude of suppression of OP amplitudes as in age-matched mice dark reared from birth. These findings demonstrate that both the normal developmental changes and the maintenance of mature inner retinal light response in adult animals require visual stimulation. These results indicate a degree of activity-dependent plasticity in mouse retina that has not been previously described.

Seeing more clearly: recent advances in understanding retinal circuitry.

Among 10 breakthroughs that Science announced at the end of 2002 was the discovery of a photosensing (melanopsin-containing) retinal ganglion cell (RGC) and its role in entraining the circadian clock. This breakthrough exemplifies the ultimate goal of neuroscience: to understand the nervous system from molecules to behavior. Light-sensing RGCs constitute one of a dozen discrete RGC populations coding various aspects of visual scenes by virtue of their unique morphology, physiology, and coverage of the retina. Interestingly, the function of the melanopsin-containing RGCs in entraining the circadian clock need not involve much retinal processing, making it the simplest form of processing in the retina. This review focuses on recent advances in our understanding of retinal circuitry, visual processing, and retinal development demonstrated by innovative experimental techniques. It also discusses the advantages of using the retina as a model system to address some of the key questions in neuroscience.

Diversity of glycine receptors in the mouse retina: localization of the alpha3 subunit.

Glycine receptors (GlyRs) and their role in retinal circuitry were analyzed immunocytochemically in wild-type and GlyR alpha3 subunit-deficient (Glra3(-/-)) mouse retinae. GlyRs are localized in the inner plexiform layer in brightly fluorescent puncta, which are likely to represent postsynaptically clustered GlyRs. Approximately one third of the clusters were found to contain the alpha1 subunit, and half possessed the alpha3 subunit. However, these two GlyR isoforms were localized at different glycinergic synapses. In the Glra3(-/-) mouse, alpha3 subunit clusters were completely eliminated, although the total number of GlyR clusters was only slightly reduced. This finding indicates that other GlyR subunits (such as alpha2 or alpha4) may have compensated for the loss of the alpha3 subunit. Characteristic expression patterns of the alpha1 and alpha3 subunits within the synaptic circuits of the retina were revealed by double labeling sections for GlyRs and markers that define specific retinal neurons. The alpha1 subunit mediates signal transfer in the rod pathway between AII amacrine cells and OFF-cone bipolar cells. In contrast, the alpha3 subunit appears to be predominantly involved with the cone pathways. Thus, expression of different GlyR alpha subunit genes correlates with anatomically defined connectivities.

Segregation of object and background motion in the retina.

An important task in vision is to detect objects moving within a stationary scene. During normal viewing this is complicated by the presence of eye movements that continually scan the image across the retina, even during fixation. To detect moving objects, the brain must distinguish local motion within the scene from the global retinal image drift due to fixational eye movements. We have found that this process begins in the retina: a subset of retinal ganglion cells responds to motion in the receptive field centre, but only if the wider surround moves with a different trajectory. This selectivity for differential motion is independent of direction, and can be explained by a model of retinal circuitry that invokes pooling over nonlinear interneurons. The suppression by global image motion is probably mediated by polyaxonal, wide-field amacrine cells with transient responses. We show how a population of ganglion cells selective for differential motion can rapidly flag moving objects, and even segregate multiple moving objects.

Zebrafish retinal slice preparation.

This paper describes the protocol for generating thin (approximately 100 microm) slices of the zebrafish retina. Retinal slices retain the cytoarchitecture and synaptic contacts found in vivo, allowing neurons to be identified prior to physiological recordings. These characteristics distinguish retinal slices from both isolated cell and eyecup preparations. Studies using the zebrafish retinal slice have classified different retinal cell types, documented voltage- and ligand-gated current responses in distal bipolar neurons, and correlated physiological responses with neuronal morphology. Data collected using this protocol have provided baseline information about retinal circuitry that can be directly applied to behavioral studies examining visual function and/or mutants with visual system defects.

Correlation of regenerable opsin with rod ERG signal in Rpe65-/- mice during development and aging.

RPE65 has been shown to be essential for the production of 11-cis retinal by the retinal pigment epithelium. Mutations in RPE65 are known to be associated with severe forms of early-onset retinal dystrophy. This project was designed to determine the amount of regenerable opsin in Rpe65-/- mice during development and aging, and to examine the function of this rhodopsin by electroretinography (ERG). Young and aged Rpe65-/- and wild-type (WT) mice were dark adapted. Endogenous rhodopsin and regenerable opsin were measured using absorption-difference spectrophotometry. Photoreceptor function was assessed with scotopic single-flash ERGs and photoreceptors were counted in histologic sections. Opsin's primary structure was analyzed by mass-spectrometric mapping. Unlike WT mice, amounts of regenerable opsin in Rpe65-/- mice decreased significantly with age, which correlated with a decrease in the number of photoreceptors and a decline in ERG amplitudes. Opsin structure, however, did not change. No endogenous levels of rhodopsin were measurable in the Rpe65-/- mice (detection limit: 0.225 pmol). 11-cis Retinal injections resulted in the regeneration of similar amounts of rhodopsin and improved rod function in a comparable way, irrespective of age. In the aged Rpe65-/- mouse, opsin levels decrease because of the loss of photoreceptors. The remaining opsin is structurally intact, and the components of the phototransduction cascade and the retinal circuitry remain functional, despite the absence of normal photoreceptor activity.

Fast and Slow Contrast Adaptation in Retinal Circuitry

The visual system adapts to the magnitude of intensity fluctuations, and this process begins in the retina. Following the switch from a low-contrast environment to one of high contrast, ganglion cell sensitivity declines in two distinct phases: a fast change occurs in <0.1 s, and a slow decrease over ∼10 s. To examine where these modulations arise, we recorded intracellularly from every major cell type in the salamander retina. Certain bipolar and amacrine cells, and all ganglion cells, adapted to contrast. Generally, these neurons showed both fast and slow adaptation. Fast effects of a contrast increase included accelerated kinetics, decreased sensitivity, and a depolarization of the baseline membrane potential. Slow adaptation did not affect kinetics, but produced a gradual hyperpolarization. This hyperpolarization can account for slow adaptation in the spiking output of ganglion cells.

IL-4 increases GABAergic phenotype in rat retinal cell cultures: involvement of muscarinic receptors and protein kinase C

Interleukin-4 (IL-4) is an anti-inflammatory cytokine. During injuries, infections and neurodegenerative diseases, high levels of this molecule are expressed in the brain. In the present work, we investigated the effect of IL-4 on GABAergic differentiation of retinal cells kept in vitro. We analyzed either the uptake of [ 3H]-γ-aminobutyric acid (GABA) or the expression of glutamic acid decarboxylase (GAD-67) following IL-4 treatment. We have also investigated the pharmacological modulation of the [ 3H]-GABA uptake by cholinergic activation. Our results demonstrate that IL-4 increases the uptake of [ 3H]-GABA after 48 h in culture in a dose-dependent manner (0.5–100 U/ml). The maximal effect was obtained with 5 U/ml (75% increase). This effect was blocked by 1 mM of nipecotic acid, demonstrating the involvement of the GAT-1 subtype of GABA transporter. The IL-4 effect depends on M1 muscarinic activity, an increase in intracellular calcium levels, tyrosine kinase activity and protein kinase C (PKC) activity. Treatment with IL-4 for 48 h induced an increase of 90% in the number of GAD- and GABA-immunoreactive cells when compared with control cultures. Our results indicate that IL-4 modulates the GABAergic phenotype of retinal cells in culture. This result can suggest an important role for this cytokine either during the normal development of retinal circuitry or during neuroprotection after injuries.

Will retinal implants restore vision?Zrenner E. Science 2002;295:1022–1025.

While there are many examples of electronic devices that support or replace the function of defective tissues-such as cochlear implants and pacemakers-restoring vision with electronic devices implanted into the retina is much more difficult. Candidates for such electronic devices are patients with loss of photoreceptor function and intact inner retinal circuits. These candidates may include patients with retinitis pigmentosa or age-related macular degeneration. The origins of electronic retinal implants started in 1956 with the placement of a selenium cell behind the retina of a blind patient. The cell enabled the patient to perceive the sensation of light. Later attempts centered on coupling electrodes to the visual cortex, although those methods were met with limited success due to limited spatial resolution. In the 1990s, researchers directed their efforts to developing an electronic prosthesis that could be directly implanted into the retina. These retinal implants are currently being developed in the United States, Germany, and Japan. There are two types of electronic retinal implants: subretinal and epiretinal. The subretinal device is implanted between the outer retina and retinal pigment epithelium. Thousands of light-sensitive microphotodiodes equipped with microelectrodes are stimulated by light passing through the retina. A small electrical current is then transmitted to retinal sensory neurons. The epiretinal implant is a readout chip that receives electrical signals containing image information from a distant camera. The epiretinal implant is placed on the retinal surface and directly stimulates ganglion cells. Weaknesses of the subretinal implant include oxidation and insufficient stimulation of adjacent retinal neurons. Weaknesses of the epiretinal implant include the need for additional data processing since the inherent retinal circuitry is not utilized and problems with fixation of the implant. Subretinal implants in humans have shown the feasibility of generating perception, although this was achieved at high charge densities and are a long way from object recognition. Results from a limited number of patients with epiretinal implants have not been released yet. There are many questions that remain unanswered, such as biocompatibility, tolerance by retinal neurons, limitations of resolution, and whether peripheral vision is compromised.—Hans E. Grossniklaus

Somatostatin (SRIF) and SRIF receptors in the mouse retina

In the retina, somatostatin (SRIF) acts as a neuromodulator by interacting with specific SRIF subtype (sst) receptors. The aim of this study was to detect mRNAs for sst 1–5 receptors by semiquantitative RT-PCR and to determine the cellular localization of either SRIF or individual SRIF receptor immunoreactivities. Size, density and absolute number of immunolabeled somata were measured using computer-assisted image analysis. With RT-PCR we found that all five sst receptor mRNAs were expressed, with highest levels of sst 2 and sst 4 receptors. SRIF immunolabeling was localized to sparse-occurring amacrine cells in the inner nuclear layer (INL) and to displaced amacrine cells in the ganglion cell layer (GCL). sst 2A receptors were localized to protein kinase- (PKC) immunoreactive (IR) rod bipolar cells, calbindin- (CaBP-) IR horizontal cells, tyrosine hydroxylase- (TH-) IR amacrine cells and glycinergic amacrine cells. None of the sst 2A-IR amacrine cells were found to express parvalbumin (PV) immunoreactivity. sst 4 receptor immunolabeling was localized to CaBP-IR and CaBP-non-IR cells in the GCL that originated long process bundles in the GC axon layer. These cells were not observed after optic nerve transection and they were therefore interpreted as ganglion cells. Quantitative analysis showed that all of the PKC-IR rod bipolar cells, CaBP-IR horizontal cells, and TH-IR amacrine cells and 5% of the glycinergic amacrine cells expressed sst 2A receptors. In addition, 4–6% of the putative ganglion cells expressed sst 4 receptors. The localization of SRIF to sparse-occurring retinal neurons, together with the widespread expression of sst 2A and sst 4 receptors suggests that SRIF acts at multiple levels of retinal circuitry. These results provide a database for investigations of the functional retinal networks in mice with genetic alterations of somatostatinergic transmission.

Light- and electron-microscopic analysis of vasoactive intestinal polypeptide-immunoreactive amacrine cells in the guinea pig retina.

Vasoactive intestinal polypeptide (VIP) is a neuroactive substance that is expressed in both nonmammalian and mammalian retinas. This study investigated the morphology and synaptic connections of VIP-containing neurons in the guinea pig retina by immunocytochemistry, by using antisera against VIP. Specific VIP immunoreactivity was localized to a population of wide-field and regularly spaced amacrine cells with processes ramifying mainly in strata 1 and 2 of the inner plexiform layer (IPL). Double-label immunohistochemistry demonstrated that all VIP-immunoreactive cells possessed gamma-aminobutyric acid immunoreactivity. The synaptic connectivity of VIP-immunoreactive amacrine cells was identified in the IPL by electron microscopy. The VIP-labeled amacrine cell processes received synaptic input from other amacrine cell processes and bipolar cell axon terminals in strata 1 to 3 of the IPL. The most frequent postsynaptic targets of VIP-immunoreactive amacrine cells were other amacrine cell processes in strata 1 to 3 of the IPL. Synaptic outputs to bipolar cells were also observed in strata 1 to 3 of the IPL. In addition, ganglion cell dendrites were also postsynaptic to VIP-immunoreactive neurons in the sublamina a of the IPL. These studies show that one type of VIP-immunoreactive amacrine cells make contact predominantly with other amacrine cell processes. This finding suggests that VIP-containing amacrine cells may influence inner retinal circuitry, thus mediating visual processing.

Light- and electron-microscopic analysis of neuropeptide Y-immunoreactive amacrine cells in the guinea pig retina.

We investigated the morphology and synaptic connections of neuropeptide Y (NPY)-containing neurons in the guinea pig retina by immunocytochemistry, using antisera against NPY. Specific NPY immunoreactivity was localized to a population of wide-field and regularly spaced amacrine cells with processes ramifying mainly in stratum 1 of the inner plexiform layer (IPL). Double-label immunohistochemistry demonstrated that all NPY-immunoreactive cells possessed glutamic acid decarboxylase 65 immunoreactivity. The synaptic connectivity of NPY-immunoreactive amacrine cells was identified in the IPL by electron microscopy. The NPY-labeled amacrine cell processes received synaptic input from other amacrine cell processes and bipolar cell axon terminals in stratum 1 of the IPL. The most frequent postsynaptic targets of NPY-immunoreactive amacrine cells were other amacrine cell processes. Synaptic outputs to bipolar cells were also observed in a small number of cases. This finding suggests that NPY-containing amacrine cells may influence inner retinal circuitry in stratum 1 of the IPL, thus mediating visual processing.

The role of early neural activity in the maturation of turtle retinal function.

In the developing vertebrate retina, ganglion cells fire spontaneous bursts of action potentials long before the eye becomes exposed to sensory experience at birth. These early bursts are synchronised between neighbouring retinal ganglion cells (RGCs), yielding unique spatiotemporal patterns: 'waves' of activity sweep across large retinal areas every few minutes. Both at retinal and extraretinal levels, these embryonic retinal waves are believed to guide the wiring of the visual system using hebbian mechanisms of synaptic strengthening. In the first part of this review, we recapitulate the evidence for a role of these embryonic spontaneous bursts of activity in shaping developing complex receptive field properties of RGCs in the turtle embryonic retina. We also discuss the role of visual experience in establishing RGC visual functions, and how spontaneous activity and visual experience interact to bring developing receptive fields to maturation. We have hypothesised that the physiological changes associated with development reflect modifications in the dendritic arbours of RGCs, the anatomical substrate of their receptive fields. We demonstrate that there is a temporal correlation between the period of receptive field expansion and that of dendritic growth. Moreover, the immature spontaneous activity contributes to dendritic growth in developing RGCs. Intracellular staining of RGCs reveals, however, that immature receptive fields only rarely show direct correlation with the layout of the corresponding dendritic tree. To investigate the possibility that not only the presence of the spontaneous activity, but even the precise spatiotemporal patterns encoded in retinal waves might contribute to the refinement of retinal neural circuitry, first we must clarify the mechanisms mediating the generation and propagation of these waves across development. In the second part of this review, we present evidence that turtle retinal waves, visualised using calcium imaging, exhibit profound changes in their spatiotemporal patterns during development. From fast waves sweeping across large retinal areas and recruiting many cells on their trajectory at early stages, waves become slower and eventually stop propagating towards hatching, when they become stationary patches of neighbouring coactive RGCs. A developmental switch from excitatory to inhibitory GABAA responses appears to mediate the modification in spontaneous activity patterns while the retina develops. Future chronic studies using specific spatiotemporal alterations of the waves will shed a new light on how the wave dynamics help in sculpting retinal receptive fields.

The Reelin Pathway Modulates the Structure and Function of Retinal Synaptic Circuitry

The formation of synaptic connections requires the coordination of specific guidance molecules and spontaneous neuronal activity. The visual system has provided a useful model for understanding the role of these cues in shaping the precise connections from the neural retina to the brain. Here, we demonstrate that two essential genes in the Reelin signaling pathway function during the patterning of synaptic connectivity in the retina. Physiological studies of mice deficient in either reelin or disabled-1 reveal an attenuation of rod-driven retinal responses. This defect is associated with a decrease in rod bipolar cell density and an abnormal distribution of processes in the inner plexiform layer . These results imply that, in addition to its essential role during neuronal migration, the Reelin pathway contributes to the formation of neuronal circuits in the central nervous system.

A Critical Role of the Strychnine-Sensitive Glycinergic System in Spontaneous Retinal Waves of the Developing Rabbit

In the developing vertebrate retina, spontaneous electric activity occurs rhythmically in the form of propagating waves and is believed to play a critical role in activity-dependent visual system development, including the establishment of precise retinal and geniculate circuitry. To elucidate how spontaneous retinal waves encode specific developmental cues at various developmental stages, it is necessary to understand how the waves are generated and regulated. Using Ca(2+) imaging and patch clamp in a flat-mount perinatal rabbit retinal preparation, this study demonstrates that, in addition to the cholinergic system, a strychnine-sensitive system in the inner retina plays an obligatory and developmentally regulated role in the initiation and propagation of spontaneous retinal waves. This system, which is believed to be the glycinergic network, provided an excitatory drive during early retinal development. It then became inhibitory after postnatal day 1 (P1) to P2, an age when a number of coordinated transitions in neurotransmitter systems occurred concomitantly, and finally contributed to the complete inhibition and disappearance of spontaneous waves after P7-P9. This glycinergic contribution was notably distinct from that of the ionotropic GABAergic system, which was found to exert an inhibitory but nonessential influence on the early wave formation. Blocking glycine- and GABA-gated anion currents had opposing effects on spontaneous retinal waves between embryonic day 29 and P0, suggesting that Cl(-) transporters, particularly R(+)-butylindazone-sensitive K-Cl cotransporters, may have a synapse- and/or cell type-specific distribution pattern, in addition to an age-dependent expression pattern in the inner retina. Overall, the results revealed an important reliance of spontaneous retinal waves on dynamic and coordinated interactions among multiple, nonredundant neurotransmitter systems.

Cell transplantation as a treatment for retinal disease.

It has been shown that photoreceptor degeneration can be limited in experimental animals by transplantation of fresh RPE to the subretinal space. There is also evidence that retinal cell transplants can be used to reconstruct retinal circuitry in dystrophic animals. Here we describe and review recent developments that highlight the necessary steps that should be taken prior to embarking on clinical trials in humans.

Modulation of potassium current and calcium influx by somatostatin in rod bipolar cells isolated from the rabbit retina via sst2 receptors.

Somatostatin (somatotropin release-inhibiting factor, SRIF) receptor subtypes are expressed by several retinal neurons, suggesting that SRIF acts at multiple levels of the retinal circuitry, although functional data on this issue are scarce. Of the SRIF receptors, the sst2A isoform is expressed by rod bipolar cells (RBCs) of the rabbit retina, and in isolated RBCs we studied the role of sst2 receptors in modulating both K+ current (IK) and the intracellular free [Ca2+] ([Ca2+]i) using both voltage-clamp and Ca2+-imaging techniques. SRIF and octreotide (a SRIF agonist that binds to sst2 receptors) inhibited that component of IK corresponding to the activation of large-conductance, Ca2+- and voltage-dependent K+ channels (IBK) and reduced the K+-induced [Ca2+]i accumulation, suggesting that SRIF effects on IBK may have been secondary to inhibition of Ca2+ channels. Octreotide effects on IBK or on [Ca2+]i accumulation were prevented by RBC treatment with L-Tyr8-Cyanamid 154806, a novel sst2 receptor antagonist, indicating that SRIF effects were mediated by sst2 receptor activation. The present data indicate that SRIF may modulate the information flow through second-order retinal neurons via an action predominantly at sst2 receptors, contribute to the proposition that SRIF be added to the growing list of retinal neuromodulators, and suggest that one of its possible roles in the retina is to regulate transmitter release from RBCs.

Vesicle-associated membrane protein isoforms in the tiger salamander retina.

Vesicle associated membrane protein (VAMP; also known as synaptobrevin) is a key component of the core complex needed for docking and fusion of synaptic vesicles with the presynaptic plasma membrane. Recent work indicates that the precise complement of presynaptic proteins associated with transmitter release and their isoforms vary among synapses, presumably conferring specific functional release properties. The retina contains two types of vesicular synapses with distinct morphologic, functional, and biochemical characteristics: ribbon and conventional synapses. Although the precise complement of presynaptic proteins is known to differ between conventional and ribbon synapses and among conventional synapses, the distribution of VAMP isoforms among retinal synapses has not been determined. The expression and localization of VAMP isoforms in the salamander retina, a major model system for studies of retinal circuitry, was examined by using immunocytochemical and immunoblotting methods. Both methods indicated that at least two VAMP isoforms were expressed in salamander retina. One isoform, recognized by an immunoglobulin M antibody that recognizes both mammalian VAMP-1 and VAMP-2, was associated with photoreceptor and bipolar cell terminals as well as many conventional synapses, and probably corresponds to mammalian VAMP-2. A different VAMP isoform associated with a subset of amacrine cells, was recognized only by antibodies directed against the N-terminus of mammalian VAMP-2. An antiserum directed against the N-terminus of mammalian VAMP-1 did not specifically recognize any salamander VAMPs in either immunocytochemical or immunoblotting experiments. Heterogeneous distribution of VAMP isoforms among conventional retinal synapses was confirmed by double labeling for synapsin I, a marker for conventional synapses. These studies indicate that VAMP isoforms are expressed heterogeneously among retinal synapses but cannot account for the differences in transmitter release characteristics at ribbon and conventional synapses. These results also corroborate previous studies in Xenopus indicating that the N-terminus of nonmammalian VAMP isoforms differs from their mammalian counterparts.