Distribution of two splice variants of the glutamate transporter GLT-1 in the developing rat retina

The distributions of a carboxyl terminal splice variant of the glutamate transporter GLT-1, referred to as GLT-1B, and the carboxyl terminus of the originally described variant of GLT-1, referred to hereafter as GLT-1 alpha, were examined using specific antisera. GLT-1B was present in the retina at very early developmental stages. Labelling was demonstrable at embryonic day 14, and strong labelling was evident by embryonic day 18. Such labelling was initially restricted to populations of cone photoreceptors, the processes of which extended through the entire thickness of the retina and appeared to make contact with the retinal ganglion cells. During postnatal development the GLT-1B-positive photoreceptor processes retracted to form the outer plexiform layer, and around postnatal day 7, GLT-1B-immunoreactive bipolar cells appeared. The pattern of labelling of bipolar cell processes within the inner plexiform layer changed during postnatal development. Two strata of strongly immunoreactive terminals were initially evident in the inner plexiform layer, but by adulthood these two bands were no longer evident and labelling was restricted to the somata and processes (but not synaptic terminals) of the bipolar cells, as well as the somata, processes, and terminals of cone photoreceptors. By contrast, GLT-1 alpha appeared late in postnatal development and was restricted mainly to a population of amacrine cells, although transient labelling was also associated with punctate elements in the outer plexiform layer, which may represent photoreceptor terminals.     

Neural differentiation and synaptogenesis in retinal development

To investigate the pattern of neural differentiation and synaptogenesis in the mouse retina, immunolabeling, Brd U assay and transmission electron microscopy were used. We show that the neuroblastic cell layer is the germinal zone for neural differentiation and retinal lamination. Ganglion cells differentiated initially at embryonic day 13(E13), and at E18 horizontal cells appeared in the neuroblastic cell layer. Neural stem cells in the outer neuroblastic cell layer differentiated into photoreceptor cells as early as postnatal day 0(P0), and neural stem cells in the inner neuroblastic cell layer differentiated into bipolar cells at P7. Synapses in the retina were mainly located in the outer and inner plexiform layers. At P7, synaptophysin immunostaining appeared in presynaptic terminals in the outer and inner plexiform layers with button-like structures. After P14, presynaptic buttons were concentrated in outer and inner plexiform layers with strong staining. These data indicate that neural differentiation and synaptogenesis in the retina play important roles in the formation of retinal neural circuitry. Our study showed that the period before P14, especially between P0 and P14, represents a critical period during retinal development. Mouse eye opening occurs during that period, suggesting that cell differentiation and synaptic formation lead to the attainment of visual function.     

Characterization of small-field bistratified amacrine cells in macaque retina labeled by antibodies against synaptotagmin-2

Macaque retinae were immunostained with monoclonal antibodies directed against the protein synaptotagmin‐2 (Syt2). Syt2 was localized in a population of small‐field amacrine cells, whose cell bodies formed a regular mosaic within the inner nuclear layer, indicating they represent a single amacrine cell type. The labeled amacrine cells had a bistratified appearance with a dense dendritic plexus in the OFF‐layer and only a few lobular processes extending into the ON‐layer of the inner plexiform layer, similar to A8 amacrine cells described in cat and human retina. Syt2‐labeled cells were immunoreactive for glycine but lacked immunoreactivity for γ‐aminobutyric acid (GABA), suggesting they use glycine as their neurotransmitter. The density of these cells increases from ～200/mm² in peripheral retina to ～1,400/mm² in central retina. Their bipolar cell input was studied by immunolabeling experiments using various bipolar cell markers combined with CtBP2, a marker of presynaptic ribbons. Our data show that Syt2‐labeled amacrine cells receive input from both OFF and ON cone bipolar cells, as well as from rod bipolar cells. The OFF input is dominated by the diffuse bipolar cell DB1 (44%) and the OFF midget bipolar cell (38%). Here we describe a population of bistratified small‐field amacrine cells closely resembling A8 amacrine cells and their cone‐dominated bipolar cell input. J. Comp. Neurol. 521:709–724, 2013. © 2012 Wiley Periodicals, Inc.     

Early steps in the assembly of photoreceptor ribbon synapses in the mouse retina: The involvement of precursor spheres

The retinal photoreceptor ribbon synapse is a chemical synapse structurally and functionally specialized for the tonic release of neurotransmitter. It is characterized by the presynaptic ribbon, an electron‐dense organelle at the active zone covered by hundreds of synaptic vesicles. In conventional synapses, dense‐core transport vesicles carrying a set of active zone proteins are implicated in early steps of synapse formation. In photoreceptor ribbon synapses, synaptic spheres are suggested to be involved in ribbon synapse assembly, but nothing is known about the molecular composition of these organelles. With light, electron, and stimulated emission depletion microscopy and immunocytochemistry, we investigated a series of presynaptic proteins during photoreceptor synaptogenesis. The cytomatrix proteins Bassoon, Piccolo, RIBEYE, and RIM1 appear early in synaptogenesis. They are transported in nonmembranous, electron‐dense, spherical transport units, which we called precursor spheres, to the future presynaptic site. Other presynaptic proteins, i.e., Munc13, CAST1, RIM2, and an L‐type Ca²⁺ channel α1 subunit are not associated with the precursor spheres. They cluster directly at the active zone some time after the first set of cytomatrix proteins has arrived. By quantitative electron microscopy, we found an inverse correlation between the numbers of spheres and synaptic ribbons in the postnatally developing photoreceptor synaptic terminals. From these results, we suggest that the precursor spheres are the transport units for proteins of the photoreceptor ribbon compartment and are involved in the assembly of mature synaptic ribbons. J. Comp. Neurol. 512:814–824, 2009. © 2008 Wiley‐Liss, Inc.     

Type 3a and type 3b OFF cone bipolar cells provide for the alternative rod pathway in the mouse retina

The mammalian retina provides several pathways to relay the information from the photoreceptors to the ganglion cells. Cones feed into ON and OFF cone bipolar cells that excite ON and OFF ganglion cells, respectively. In the "classical" rod pathway, rods feed into rod bipolar cells that provide input to both the ON and the OFF pathway via AII amacrine cells. Recent evidence suggests an alternative rod pathway in which rods directly contact some types of OFF cone bipolar cells. The mouse has become an important model system for retinal research. We performed an immunohistochemical analysis on the level of light and electron microscopy to identify the bipolar cells and ganglion cells that are involved in the alternative rod pathway of the mouse retina. 1) We identify a new bipolar cell type, showing that type 3 OFF cone bipolar cells comprise two distinct cell types, that we termed 3a and 3b. Type 3a cells express the ion channel HCN4. Type 3b bipolar cells represent a hitherto unknown cell type that can be identified with antibodies against the regulatory subunit RIIbeta of protein kinase A. 2) We show that both 3a and 3b cells form flat contacts at cone pedicles and rod spherules. 3) Finally, we identify an OFF ganglion cell type whose dendrites costratify with type 3a and 3b bipolar cell axon terminals. These newly identified cell types represent the basis of a neuronal circuit in the mammalian retina that could provide for an alternative fast rod pathway.     

Spatiotemporal coordination of rod and cone photoreceptor differentiation in goldfish retina

In this study, we have compared spatial and temporal aspects of development of new rods and cones in the adult goldfish by using a combination of bromodeoxyuridine immunocytochemistry and opsin in situ hybridization to determine the intervals between terminal mitosis (cell “birth”) and expression of opsin mRNA for each photoreceptor cell type. The goldfish opsins include rod opsin and four different cone opsins: red, green, blue, and ultraviolet. In a cohort of photoreceptors born at the same time, rods expressed opsin mRNA within 3 days of cell birth, while expression of cone opsin mRNA required at least 7 days. This temporal discrepancy in differentiation, coupled with a discordance in the site of cell genesis of rods and cones, allowed opsin expression to commence in both cell types in approximately the same retinal location. Commitment to the generic cone phenotype occurred within approximately 6 days throughout the cone cohort, as indicated by expression of interphotoreceptor retinoid-binding protein (IRBP) mRNA, but expression of a specific spectral phenotype was delayed until rods differentiated nearby. Onset of expression of cone opsin mRNA followed a phenotype-specific sequence: red, then green, then blue, and finally ultraviolet; in situ hybridization with two opsin probes confirmed that individual photoreceptors expressed only one type of opsin as they differentiated. This stepwise process of cone differentiation is consistent with the hypothesis that cell-cell interactions among developing photoreceptors may coordinate selection of specific photoreceptor phenotypes. J. Comp. Neurol. 382:272-284, 1997. © 1997 Wiley-Liss, Inc.     

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.     

Differential distribution and developmental expression of synaptic vesicle protein 2 isoforms in the mouse retina

Synaptic vesicle protein 2 (SV2), a ubiquitous synaptic vesicle protein, is known to participate in the regulation of Ca(2+)-mediated synaptic transmission, although its precise function has not been established. Three SV2 isoforms (SV2A, SV2B, SV2C) have been identified recently, each of which has a unique distribution in brain, suggesting synapse-specific functions. To determine if SV2A, -B, and -C are differentially distributed among synapses in the retina and the sequence of their development, we examined their distribution and expression patterns immunocytochemically in adult and developing mouse retina. The three SV2 isoforms were differentially distributed in the synapses of the two plexiform layers in the adult retina. SV2A was present in cone, but not rod, terminals in the outer plexiform layer (OPL) and in many synaptic terminals in the inner plexiform layer (IPL). SV2B was present only in the ribbon synapse-containing terminals of rod and cone photoreceptors and bipolar cells. SV2C was present in starburst amacrine cells, other conventional synapses in the IPL of unknown origin, and in presumptive interplexiform cell terminals in the INL and OPL. Each SV2 isoform was expressed in its distinct presynaptic terminals early and throughout postnatal development. In addition, SV2A was transiently expressed by developing horizontal cells. The unique distribution of each isoform suggests potentially distinct functions at different types of synapses, with SV2B having ribbon synapse-specific functions, and SV2C being important for the functions of starburst amacrine cells. Rod and cone terminals contain different complements of SV2 isoforms, indicating that ribbon synapses are not all identical. The early expression of SV2 isoforms prior to initiation of synapse formation suggests that they may have important synapse-specific roles during synaptogenesis.     

The immunocytochemical localization of connexin 36 at rod and cone gap junctions in the guinea pig retina

Connexin 36 (Cx36) is a channel-forming protein found in the membranes of apposed cells, forming the hexameric hemichannels of intercellular gap junction channels. It localizes to certain neurons in various regions of the brain including the retina. We characterized the expression pattern of neuronal Cx36 in the guinea pig retina by immunocytochemistry using specific antisera against Cx36 and green/red cone opsin or recoverin. Strong Cx36 immunoreactivity was visible in the ON sublamina of the inner plexiform layer and in the outer plexiform layer, as punctate labelling patterns. Double-labelling experiments with antibody directed against Cx36 and green/red cone opsin or recoverin showed that strong clustered Cx36 immunoreactivity localized to the axon terminals of cone or close to rod photoreceptors. By electron microscopy, Cx36 immunoreactivity was visible in the gap junctions as well as in the cytoplasmic matrices of both sides of cone photoreceptors. In the gap junctions between cone and rod photoreceptors, Cx36 immunoreactivity was only visible in the cytoplasmic matrices of cone photoreceptors. These results clearly indicate that Cx36 forms homologous gap junctions between neighbouring cone photoreceptors, and forms heterologous gap junctions between cone and rod photoreceptors in guinea pig retina. This focal location of Cx36 at the terminals of the photoreceptor suggests that rod photoreceptors can transmit rod signals to the pedicle of a neighbouring cone photoreceptor via Cx36, and that the cone in turn signals to corresponding ganglion cells via ON and OFF cone bipolar cells.     

Distribution of soluble guanylyl cyclase in rat retina

The nitric oxide (NO)-cGMP pathway is implicated in modulation of visual information processing in the retina. Despite numerous functional studies of this pathway, information about the retinal distribution of the major downstream effector of NO, soluble guanylyl cyclase (sGC), is very limited. In the present work, we have used immunohistochemistry and multiple labeling to determine the distribution of sGC in rat retina. sGC was present at high levels in inner retina but barely detectable in outer retina. Photoreceptors and horizontal cells, as well as Müller cells, were immunonegative, whereas retinal ganglion cells exhibited moderate staining for sGC. Strong immunostaining was found in subpopulations of bipolar and amacrine cells, but staining was weak in rod bipolar cells, and AII amacrine cells were immunonegative. Double labeling of sGC with neuronal nitric oxide synthase showed that the two proteins are generally located in adjacent puncta in inner plexiform layer, implying paracrine interactions. Our results suggest that the NO-cGMP pathway modulates the neural circuitry in inner retina, preferentially within the cone pathway.     

Spatial and temporal patterns of growth and differentiation of cone oil droplets in the chick retina

Avian cone photoreceptors have an oil droplet in the outer portion of their inner segment that acts as a long-pass cut-off filter between incident light and visual pigment. Chick cone droplets are mainly red, orange, yellow, green, and colorless, and the colors are due to three carotenoid pigments with characteristic absorption spectra. Little is known of the differentiation of this organelle, the natural marker of cones, and the little that is known is largely controversial. We used flat whole-mounts of fresh retinas to study the time and place of the appearance of droplets, their growth rates, the sequence of droplet color differentiation, and the spatial distribution of these colors. We show that droplet differentiation starts on embryonic Day 10 (E10) in a relatively small area above the optic nerve head. The differentiation spreads to the rest of the retina in a manner similar to that of photoreceptor neurogenesis, with three decreasing gradients of droplet size and color between E13-E20: from central to peripheral, dorsal to ventral, and temporal to nasal. The rate of growth of the droplets was not constant, but showed a maximum between E17 and postnatal Day 1 (P1) in most of the retinal zones. Color differentiation started at E16-E17, 5-6 days after their appearance, when the droplets were already of considerable size. Initially, all droplets were colorless, and then turned pale green or yellow to acquire progressively the mature colors. Differentiation ended in the whole retina by P15, with ventral droplets of larger diameter than dorsal ones, the peripheral ones generally larger than the central ones, and with the color distribution varying with the retinal area. Our results show that growth and color differentiation of the droplets is regulated temporally and spatially, and the cones complete differentiation at P15 rather than at prenatal stages, as is thought generally.     

Cellular degeneration and synaptogenesis in the developing retina of the rat

We have investigated the time course and magnitude of cellular degeneration in the ganglion cell layer and the presumptive amacrine and bipolar regions of the inner nuclear layer during the development of the retina in the rat. Pyknotic profiles are present in the ganglion cell layer during the first 2 postnatal weeks, reaching peak numbers during the first 4 postnatal days (corresponding to the time of greatest loss of ganglion cells and their axons: Potts et al., '82; Lam et al., '82; Perry et al., '83). Two observations suggest that the majority of pyknotic profiles present in the ganglion cell layer during the second postnatal week are not ganglion cells. First, following injection of kainic acid into one superior colliculus, degenerating ganglion cells in the contralateral retina are cleared within 24-48 hours. Therefore, since most ganglion cell and axon loss occurs within the first postnatal week, few of the pyknotic profiles present in the second week are likely to be ganglion cells. Second, the time course of cellular degeneration in the ganglion cell layer during the second postnatal week follows a very similar pattern to that seen in the presumptive amacrine sublayer of the inner nuclear layer. Such a correspondence suggests that two phases of cell death occur in the ganglion cell layer: during the first postnatal week the majority of dying cells are ganglion cells, and in the second, most cell death is due to a loss of displaced amacrine cells. In the inner nuclear layer pyknotic profiles are most numerous in the presumptive amacrine region on postnatal days 6 and 7, and in the presumptive bipolar region on day 10. Synaptogenesis in the inner plexiform layer occurs later but reflects the order of cell death. Thus, conventional (presumed amacrine) synapses were first observed on day 11 and synaptic ribbons (indicative of bipolar synapses) on day 13. These observations suggest that amacrine and bipolar cells initiate synapses only after their numbers have stabilized.     

Antibody to calretinin stains AII amacrine cells in the rabbit retina: Double-label and confocal analyses

The AII or rod amacrine cell is a critical interneuron in the rod pathway of mammalian retinae. In this report, it is shown that commercially available antibodies to the calcium binding protein calretinin may be used to label the population of AII amacrine cells selectively. Calretinin-positive amacrine cells had the morphological attributes of AII amacrine cells. Double-labeling procedures showed that calretinin-positive somata were surrounded by dopaminergic varicosities and that calretinin-positive dendrites enclosed rod bipolar terminals, both as previously described for AII amacrine cells. By analyzing the surrounding kernel for each labeled pixel in the rod bipolar image, it is shown here that AII processes are adjacent to rod bipolar terminals at a level that far exceeds the random overlap present in images in which one label was rotated out of phase. Such a spatial relationship is indicative of synaptic connections, as well described for rod bipolar input to AII amacrine cells. AII amacrine cells also were double-labeled for calretinin and parvalbumin; however, a scattergram analysis of red versus green intensity showed that the parvalbumin antibody stained additional unidentified amacrine cells. In conclusion, at the appropriate dilution, calretinin antibodies are a useful marker for AII amacrine cells in the rabbit retina. J. Comp. Neurol. 411:3–18, 1999. © 1999 Wiley-Liss, Inc.     

Glutamatergic input is coded by spike frequency at the soma and proximal dendrite of AII amacrine cells in the mouse retina

In the mammalian retina, AII amacrine cells play a crucial role in scotopic vision. They transfer rod signals from rod bipolar cells to the cone circuit, and divide these signals into the ON and OFF pathways at the discrete synaptic layers. AII amacrine cells have been reported to generate tetrodotoxin (TTX)-sensitive repetitive spikes of small amplitude. To investigate the properties of the spikes, we performed whole-cell patch-clamping of AII amacrine cells in mouse retinal slices. The spike frequency increased in proportion to the concentration of glutamate puffer-applied to the arboreal dendrite and to the intensity of the depolarizing current injection. The spike activity was suppressed by L-2-amino-4-phosphonobutyric acid, a glutamate analogue that hyperpolarizes rod bipolar cells, puffer-applied to the outer plexiform layer. Therefore, it is most likely that the spike frequency generated by AII amacrine cells is dependent on the excitatory glutamatergic input from rod bipolar cells. Gap junction blockers reduced the range of intensity of input with which spike frequency varies. Application of TTX to the soma and the proximal dendrite of AII amacrine cells blocked the voltage-gated Na(+) current significantly more than application to the arboreal dendrite, indicating that the Na(+) channels are mainly localized in these regions. Our results suggest that the intensity of the glutamatergic input from rod bipolar cells is coded by the spike frequency at the soma and the proximal dendrite of AII amacrine cells, raising the possibility that the spikes could contribute to the OFF pathway to enhance release of neurotransmitter.     

Diurnal and circadian variation of protein kinase C immunoreactivity in the rat retina

We studied the dependence of the expression of protein kinase C immunoreactivity (PKC-IR) in the rat retina on the light:dark (LD) cycle and on circadian rhythmicity in complete darkness (DD). Two anti-PKC alpha antibodies were employed: One, which we call PKCalphabeta recognized the hinge region; the other, here termed PKCalpha, recognized the regulatory region of the molecule. Western blots showed that both anti-PKC antibodies stained an identical single band at approximately 80 kD. The retinal neurons showing PKC-IR were rod bipolar cells and a variety of amacrine neurons. After 3 weeks on an LD cycle, PKCalphabeta-IR in both rod bipolar and certain amacrine cells manifested a clear rhythm with a peak at zeitgeber time (ZT) of 06-10 hours and a minimum at ZT 18. No rhythm in total PKC-IR was observed when using the PKCalpha antibody, but, at ZT 06-10 hours, rod bipolar axon terminals showed increased immunostaining. After 48 hours in DD, with either antibody, rod bipolar cells showed increased PKC-IR. The PKCalpha antibody alone revealed that, after 48 hours, AII amacrine neurons, which lacked PKC-IR in an LD cycle, manifested marked PKC-IR, which became stronger after 72 hours. Light administered early in the dark period greatly increased PKCalphabeta-IR in rod bipolar and some amacrine neurons. Our data indicate that light and darkness exert a strong regulatory influence on PKC synthesis, activation, and transport in retinal neurons.     

Protein kinase C immunoreactivity in the pigmented and albino rat retina

The albino retina is abnormal. The central region is under-developed and some cell populations are reduced or increased in number. Not least of these anomalies is the deficit in the rod population in hypopigmented rodents and carnivores. Given this abnormality we have examined the distribution of rod bipolar cells in albino rats to determine whether this subsequent stage in the rod pathway is similarly disrupted. A monoclonal antibody to protein kinase C was used to determine the distribution of rod bipolar cells in juvenile and adult pigmented and albino rats. Immunoreactive rod bipolar cells and their processes were counted in transverse sections passing through both the central and peripheral retina. The mean densities of immunoreactive cells were significantly reduced in albino retinas at both juvenile (postnatal day 15) and adult stages, in the former by 14% and the latter by 9%. This was evident across the entire central-to-peripheral extent of the retina. The reduced rod photoreceptor population found in albinos appears therefore to be consequential for the magnitude of their major target population, rod bipolar cells. The decrease in the rod bipolar population indicates a change in retinal cytoarchitecture and implies a disruption of functional organization of the albino retina, especially that underlying the scotopic channel. This, coupled with observations that some other retinal interneuronal populations may be disrupted, implies disordered retinal processing in albinos and emphasizes the likelihood that abnormal visual function in albinos may be as much a result of anomalous retinal circuitry as of the known photoreceptor deficit or chiasmatic misrouting.     

Synaptogenesis and synaptic protein localization in the postnatal development of rod bipolar cell dendrites in mouse retina

Retinal responses to photons originate in rod photoreceptors and are transmitted to the ganglion cell output of the retina through the primary rod bipolar pathway. At the first synapse of this pathway, input from multiple rods is pooled into individual rod bipolar cells. This architecture is called convergence. Convergence serves to improve sensitivity of rod vision when photons are sparse. Establishment of convergence depends on the development of a proper complement of dendritic tips and transduction proteins in rod bipolar cells. How the dendrites of rod bipolar cells develop and contact the appropriate number of rods is unknown. To answer this question we visualized individual rod bipolar cells in mouse retina during postnatal development and quantified the number of dendritic tips, as well as the expression of transduction proteins within dendrites. Our findings show that the number of dendritic tips in rod bipolar cells increases monotonically during development. The number of tips at P21, P30, and P82 exceeds the previously reported rod convergence ratios, and the majority of these tips are proximal to a presynaptic rod release site, suggesting more rods provide input to a rod bipolar cell. We also show that dendritic transduction cascade members mGluR6 and TRPM1 appear in tips with different timelines. These finding suggest that (a) rod bipolar cell dendrites elaborate without pruning during development, (b) the convergence ratio between rods and rod bipolar cells may be higher than previously reported, and (c) mGluR6 and TRPM1 are trafficked independently during development.