Type 4 OFF cone bipolar cells of the mouse retina express calsenilin and contact cones as well as rods

Immunocytochemical discrimination of distinct bipolar cell types in the mouse retina is a prerequisite for analyzing retinal circuitry in wild-type and transgenic mice. Here we demonstrate that among the more than 10 anatomically defined mouse bipolar cell types, type 4 bipolar cells are specifically recognized by anti-calsenilin antibodies. Axon terminals in the inner plexiform layer are not readily identifiable because calsenilin is also expressed in a subset of amacrine and ganglion cells. In contrast, in the outer plexiform layer calsenilin immunoreactivity allows the analysis of photoreceptor to type 4 bipolar cell contacts. A dense plexus of calsenilin-positive dendrites makes several basal contacts at cone pedicles. An individual calsenilin-positive bipolar cell contacts five to seven cones. In addition, some calsenilin-positive dendrites contact rod photoreceptors. On average we counted 10 rod spherule contacts per type 4 bipolar cell, and approximately 10% of rods contacted type 4 bipolar cells. We suggest that type 4 bipolar cells, together with the recently described type 3a and b cells, provide an alternative and direct route from rods to OFF cone bipolar cells. In the Bassoon DeltaEx4/5 mouse, a mouse mutant that shows extensive remodeling of the rod system including sprouting of horizontal and rod bipolar cells into the outer nuclear layer due to impaired synaptic transmission, we found that in addition mixed-input (type 3 and 4) OFF bipolar cells sprout to ectopic sites. In contrast, true cone-selective type 1 and 2 OFF cone bipolar cells did not show sprouting in the Bassoon mouse mutant.     

Glycinergic contacts in the outer plexiform layer of the Xenopus laevis retina characterized by antibodies to glycine, GABA and glycine receptors

Electrophysiological experiments have predicted a direct synaptic input from glycinergic interplexiform cells (IPCs) to GABAergic horizontal cells in the Xenopus retina. However, previous ultrastructural studies failed to demonstrate this input. Here, we used three immunocytochemical approaches to investigate this issue. First, double-label postembedding immunocytochemistry with GABA- and glycine-like immunoreactivity (GABA-LI and glycine-LI) was used to study possible interactions of the glycinergic IPC with GABAergic horizontal cells. Processes postsynaptic to glycine-LI IPC terminals in the outer plexiform layer (OPL) fell into two groups, small microtubule-filled processes and larger electron-lucent processes with sparse microtubules and occasional mitochondria. In no case did we find glycine-LI synapses onto GABA-LI cells or processes. Second, pre-embedding immunocytochemistry was used to label GABA-LI cells and processes in the OPL. GABA-LI was sparse in horizontal cell axons and more intense in horizontal cell somas and in small processes. In agreement with our first set of experiments, GABA-LI profiles did not receive input from conventional synapses. Third, we localized glycine-receptor-like immunoreactivity (GlyR-LI) to several types of apparent synapses in the OPL. As expected, it was found at IPC synapses. Unexpectedly, GlyR-LI was also subsynaptic at photoreceptor synapses onto second order neurons, both at ribbon and basal junction type synapses. At least some of the GlyR-LI photoreceptor synapses were from cones. Also, GlyR-LI was apposed to photoreceptors and to unidentified small diameter processes, where no other indication of synaptic input was evident. Because glycine-LI is not found in photoreceptors, we suggest that glycine receptors at photoreceptor synapses are stimulated by glycine that diffuses from other sites, possibly from IPCs. This interpretation is consistent with available physiological studies of glycinergic effects in this retina.     

Distribution of muscarinic acetylcholine receptors on processes of isolated retinal cells

Binding of propylbenzilylcholine mustard, a muscarinic acetylcholine receptor antagonist, to isolated retinal cells was examined with light microscopic autoradiography. Dissociation of the adult tiger salamander retina yielded identifiable rod, cone, horizontal, bipolar, amacrine/ganglion, and Müller cells. Preservation of fine structure was assessed with conventional electron microscopy. For all cell types, the plasmalemma was intact and free of adhering debris; in addition, presynaptic ribbon complexes were present in photoreceptor and bipolar axon terminals indicating that synaptic structures were retained. Specific binding to cell bodies and processes was analyzed separately by using morphometric and statistical techniques. The highest grain densities occurred on processes of amacrine/ganglion cells and axons and 2 degrees and 3 degrees dendrites of bipolar neurons. Bipolar cells, however, seemed to be a heterogeneous population because there was great variation in the density of binding sites on both their axons and distal dendrites. Intermediate levels of binding were found on bipolar 1 degree dendrites and horizontal cells. No specific binding was detected on Müller cells and most parts of photoreceptors. Comparisons between cells showed that grain densities were similar for bipolar axons and amacrine/ganglion cell processes but bipolar dendrites were richer in binding sites than horizontal cell dendrites. Thus, muscarinic receptors in the salamander retina are located on amacrine/ganglion, bipolar, and horizontal cells and primarily confined to the processes which compose the two synaptic layers. In the inner plexiform layer, muscarinic receptors reside on processes from all three inner retinal neurons: in the outer synaptic layer, receptors are only on second-order cells and are more numerous along bipolar than horizontal cell dendrites.     

Subcellular distribution of connexin45 in OFF bipolar cells of the mouse retina

In the mouse retina, connexin45 (Cx45) participates in the gap junction between ON cone bipolar cells and AII amacrine cells, which constitutes an essential element of the primary rod pathway. Although it has been shown that Cx45 is also expressed in OFF bipolar cells, its subcellular localization and functional role in these cells are unknown. Here, we analyzed the localization of Cx45 on OFF bipolar cells in the mouse retina. For this, we used wild-type mice and a transgenic mouse line that expressed, in addition to native Cx45, a fusion protein consisting of Cx45 and the enhanced green fluorescent protein (EGFP). Cx45-EGFP expression generates an EGFP signal at gap junctions containing Cx45. Combining immunohistochemistry with intracellular injections, we found that Cx45 was present on dendrites and axon terminals of all OFF bipolar cell types. Cx45 was not found at intersections of two terminal processes of the same type, suggesting that Cx45 might not form gap junctions between axon terminals of the same OFF bipolar cell type but rather might connect OFF bipolar cells to amacrine or ganglion cells. In OFF bipolar cell dendrites, Cx45 was found predominantly in the proximal outer plexiform layer (OPL), well below the cone pedicles. Cx45 did not colocalize with Cx36, which is found predominantly in the distal OPL. We conclude that Cx45 is expressed on OFF bipolar cell dendrites, presumably forming gap junctions with cells of the same type, and on OFF bipolar cell axon terminals, presumably forming heterologous gap junctions with other retinal neurons.     

Selective synaptic connections in the retinal pathway for night vision

The mammalian retina encodes visual information in dim light using rod photoreceptors and a specialized circuit: rods→rod bipolar cells→AII amacrine cell. The AII amacrine cell uses sign-conserving electrical synapses to modulate ON cone bipolar cell terminals and sign-inverting chemical (glycinergic) synapses to modulate OFF cone cell bipolar terminals; these ON and OFF cone bipolar terminals then drive the output neurons, retinal ganglion cells (RGCs), following light increments and decrements, respectively. The AII amacrine cell also makes direct glycinergic synapses with certain RGCs, but it is not well established how many types receive this direct AII input. Here, we investigated functional AII amacrine→RGC synaptic connections in the retina of the guinea pig (Cavia porcellus) by recording inhibitory currents from RGCs in the presence of ionotropic glutamate receptor (iGluR) antagonists. This condition isolates a specific pathway through the AII amacrine cell that does not require iGluRs: cone→ON cone bipolar cell→AII amacrine cell→RGC. These recordings show that AII amacrine cells make direct synapses with OFF Alpha, OFF Delta and a smaller OFF transient RGC type that co-stratifies with OFF Alpha cells. However, AII amacrine cells avoid making synapses with numerous RGC types that co-stratify with the connected RGCs. Selective AII connections ensure that a privileged minority of RGC types receives direct input from the night-vision pathway, independent from OFF bipolar cell activity. Furthermore, these results illustrate the specificity of retinal connections, which cannot be predicted solely by co-stratification of dendrites and axons within the inner plexiform layer.     

Ablation of retinal horizontal cells from adult mice leads to rod degeneration and remodeling in the outer retina

In the brain, including the retina, interneurons show an enormous structural and functional diversity. Retinal horizontal cells represent a class of interneurons that form triad synapses with photoreceptors and ON bipolar cells. At this first retinal synapse, horizontal cells modulate signal transmission from photoreceptors to bipolar cells by feedback and feedforward inhibition. To test how the fully developed retina reacts to the specific loss of horizontal cells, these interneurons were specifically ablated from adult mice using the diphtheria toxin (DT)/DT-receptor system and the connexin57 promoter. Following ablation, the retinal network responded with extensive remodeling: rods retracted their axons from the outer plexiform layer and partially degenerated, whereas cones survived. Cone pedicles remained in the outer plexiform layer and preserved synaptic contacts with OFF but not with ON bipolar cells. Consistently, the retinal ON pathway was impaired, leading to reduced amplitudes and prolonged latencies in electroretinograms. However, ganglion cell responses showed only slight changes in time course, presumably because ON bipolar cells formed multiple ectopic synapses with photoreceptors, and visual performance, assessed with an optomotor system, was only mildly affected. Thus, the loss of an entire interneuron class can be largely compensated even by the adult retinal network.     

Mosaic deletion of Rb arrests rod differentiation and stimulates ectopic synaptogenesis in the mouse retina

The retinoblastoma gene (Rb) regulates neural progenitor cell proliferation and cell fate specification and differentiation. For the developing mouse retina, two distinct functions of Rb have been described: regulation of retinal progenitor cell proliferation and rod photoreceptor development. Cells that would normally become rods fail to mature and remain as immature cells in the outer nuclear layer in the adult. By using Chx10-Cre;Rb(Lox/-) mice, we generated a chimeric retina with alternating apical-basal stripes of wild-type and Rb-deficient tissue. This provides a unique model with which to study synaptogenesis at the outer plexiform layer within regions that lack differentiated rods. In regions where rods failed to differentiate, the outer plexiform layer (OPL) was disrupted. Horizontal cells formed, and their somata were appropriately aligned, but their neurites did not project laterally. Instead many horizonal cell neurites extended apically, forming ectopic synapses with photoreceptors at all levels of the outer nuclear layer. These ectopic photoreceptor terminals contained synaptic ribbons, horizontal cell processes with synaptic vesicles, and a single mitochrondrion characteristic of rod spherules. Rb-deficient bipolar cells differentiated normally, extended dendrites to the OPL, and formed synapses that were indistinguishable from adjacent wild-type cells. In contrast to OPL-positioned synapses, ectopic synapses did not contain bipolar dendrites. This finding suggests that horizontal cells and photoreceptors can form stable synapses that are devoid of bipolar dendrites outside the normal boundaries of the OPL. Finally, analysis of P4, P7, P12, and P15 retinae suggests that the apical horizontal cell processes result from their failure to establish their normal lateral projections during development.     

Localization of GABAA receptor subunits alpha 1, alpha 3, beta 1, beta 2/3, gamma 1, and gamma 2 in the salamander retina

Electrophysiological studies have demonstrated that gamma-aminobutyric acid receptors type A (GABA(A)) mediate important information processing in the retinas of salamander and other vertebrates. The pharmacology and physiology of GABA(A) receptors depend on their subunit composition. We studied the localization of GABA(A) receptor subunit isoforms alpha(1), alpha(3), beta(1), beta(2/3) (antibody BD-17 and 62-3G1), gamma(1), and gamma(2) in salamander retina with immunocytochemical methods. All three beta-subunit antibodies labeled similarly in the outer retina, especially the inner segments and synaptic terminals of rod photoreceptors (identified with protein kinase C). Somatic labeling was observed in cell bodies of some horizontal cells, bipolar cells, amacrine cells, and cells in the ganglion cell layer (GCL). Puncta were present throughout the inner plexiform layer (IPL) for beta(1) and 62-3G1, but not for BD-17. alpha(1)-immunoreactivity (IR) stained a population of presumed OFF rod-dominated bipolar cells, including dendrites, soma, and axon terminals in the distal IPL. A subtype of GABAergic amacrine cell also expressed alpha(1)-IR, with puncta sparsely distributed at the distal and proximal margins of the IPL. Both the OPL and IPL were labeled throughout for alpha(3)-IR, as opposed to the narrow distribution of alpha(1)-IR in the IPL, suggesting that the two alpha-subunits are localized at different synaptic sites. Punctate gamma(1)-IR was observed in the OPL and IPL, whereas gamma(2) was most prominent in cone photoreceptors (identified with calbindin), including the terminal telodendria, in cell bodies of some horizontal cells, amacrine cells, cells in the GCL, and less intensely in the IPL. In addition, several subunits were present in Müller cells. The differential labeling suggests the existence of GABA(A) receptor subtypes with different subunit compositions that mediate multiple GABAergic functions in salamander retina.     

Immunocytochemical localization of kainate-selective glutamate receptor subunits GluR5, GluR6, and GluR7 in the cat retina

Localizations of the kainate-selective glutamate receptor subunits GluR5, 6, and 7 were studied in the cat retina by light and electron microscopic immunocytochemistry. GluR5 immunoreactivity was observed in the cell bodies and dendrites of numerous cone bipolar cells and ganglion cells. The labeled cone bipolar cells make basal or flat contacts with cone pedicles in the outer plexiform layer, leading to their identification as OFF-center bipolar cells. Reaction product within the inner plexiform layer was observed in processes of ganglion cells at their sites of input from cone bipolar cells. Staining for GluR6 was localized to A- and B-type horizontal cells, numerous amacrine cells, and an occasional cone bipolar cell. The larger ganglion cells were also immunoreactive. As with other GluR molecules, labeling was usually confined to one of the two postsynaptic elements at a cone bipolar dyad contact. Immunoreactivity for GluR7 was very limited and was seen only in a few amacrine and displaced amacrine cells. Findings of this study are consistent with a major role for kainate receptors in mediating OFF pathways in the outer retina with participation in both OFF and ON pathways in the inner retina.     

Presynaptic and postsynaptic localization of GABAB receptors in neurons of the rat retina

The recently cloned GABA_B receptors were localized in rat retina using specific antisera. Immunolabelling was detected in the inner and outer plexiform layers (IPL, OPL), and in a number of cells in the inner nuclear layer and the ganglion cell layer. Double-labelling experiments for GABA (γ-aminobutyric acid) and GABA_B receptors, respectively, demonstrated a co-localization in horizontal cells and amacrine cells. Electron microscopy showed that GABA_B receptors of the OPL were localized presynaptically in horizontal cell processes invaginating into photoreceptor terminals. In the IPL, GABA_B receptors were present presynaptically in amacrine cells, as well as postsynaptically in amacrine and ganglion cells. The postnatal development of GABA_B receptors was also studied, and immunoreactivity was observed well before morphological and synaptic differentiation of retinal neurons. The present results suggest a presynaptic (autoreceptor) as well as postsynaptic role for GABA_B receptors. In addition, the extrasynaptic localization of GABA_B receptors could indicate a paracrine function of GABA in the retina.     

The synaptic ultrastructure in the outer plexiform layer of the catfish retina: a three-dimensional study with HVEM and conventional EM of Golgi-impregnated bipolar and horizontal cells

Synaptic structures between receptors and horizontal and bipolar cells in the outer plexiform layer (OPL) of Golgi-impregnated catfish retina were examined by conventional electron microscopy of serial ultrathin sections and by high-voltage electron microscopy (HVEM) of thick sections. Cone terminals contained multiple synaptic ribbons and rod terminals contained single synaptic ribbons. This observation was used to identify these two types of photoreceptors. The cone horizontal cell, located in the most distal part of the inner nuclear layer (INL), invaginated only cone terminals, whereas the rod horizontal cell, located in the proximal part of the INL, invaginated only rod terminals. Both lateral elements of the triad in the rod terminal originated from a single rod horizontal cell whereas the same structures in the cone terminal were often derived from several cone horizontal cells. Golgi-impregnated catfish bipolar cells were classified into two types based on the differences in their axonal arborization as described by Famiglietti et al. ('77). Axonal endings of type a bipolar cells were located in the distal part, sublamina a, of the inner plexiform layer (IPL), and axonal endings of type b cells were located in the proximal part, sublamina b, of the IPL. Dendrites from type a bipolar cells made direct contact with the synaptic ribbons in both rod and cone terminals whereas those from type b cells made indirect contact with the ribbons in both rod and cone terminals, but rare direct contact with the ribbon in rod terminals were also seen. In addition, bipolar cells made basal junctions or superficial contacts in both rod and cone terminals. The "lateral" processes of bipolar cells invaginating rods penetrated between the rod terminal and rod horizontal cell processes, and made basal junctions with both rod terminals and rod horizontal cells. There was no definitive morphological feature that could be associated with sign-conserving and sign-inverting signal transmission.     

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.     

Localization of metabotropic glutamate receptors mGluR1alpha and mGluR2/3 in the cat retina.

The distribution of metabotropic glutamate receptors 1alpha (mGluR1alpha) and mGluR2/3 in the cat retina was studied through the use of preembedding immunocytochemistry for light and electron microscopy. Staining for mGluR1alpha in the outer plexiform layer was seen in numerous punctate structures that were identified as rod spherules. Cone pedicles remained unlabeled. A number of amacrine and ganglion cell somata also were stained with processes ramifying throughout the inner plexiform layer. These processes were postsynaptic to cone bipolar cells in both sublaminae, where they comprised one but not both of the postsynaptic elements at dyad contacts. Immunostaining for mGluR2/3 was observed in horizontal cells as well as in numerous amacrine and displaced amacrine cells. Labeled amacrine processes were postsynaptic to cone bipolar cells in both sublaminae but, similar to mGluR1alpha, comprised only one of the postsynaptic elements. Staining for mGluR2/3 also was seen in amacrine processes postsynaptic to rod bipolar terminals; these processes were identified as belonging to type A17 amacrine cells. The distribution patterns indicate that both mGluR1alpha and mGluR2/3 are positioned for postsynaptic function, whereas mGluR1alpha also may contribute to the presynaptic regulation of glutamate release from rod photoreceptors.     

Localization of aspartate-like immunoreactivity in the retina of the turtle (Pseudemys scripta).

Aspartate has been reported to be a putative excitatory neurotransmitter in the retina, but little detailed information is available concerning its anatomical distribution. We used an antiserum directed against an aspartate-albumin conjugate to analyze the anatomy, dendritic stratification, and regional distribution of cell types with aspartate-like immunoreactivity in the turtle retina. The results showed dramatic differences in immunoreactivity in the peripheral versus the central retina. Strong aspartate-like immunoreactivity was shown in the peripheral retina, with many well-labeled processes in the inner plexiform layer. Many bipolar, horizontal, amacrine, and ganglion cells, some photoreceptors, and some unidentified cells were strongly immunoreactive in the peripheral retina. In contrast, although the central retina showed well-labeled horizontal cells, there was only light labeling in the inner plexiform layer with weakly immunoreactive amacrine and ganglion cells and no labeled bipolar cells. There were several strongly immunoreactive efferent nerve fibers which left the optic nerve head and arborized extensively in the retina. At the electron microscopic level, electron-dense reaction product was associated with synaptic vesicles at bipolar and amacrine cell synapses in the inner plexiform layer. These results suggest that aspartate may be involved in many diverse synaptic interactions in both the outer plexiform layer and the inner plexiform layer of the turtle retina.     

The absence of Complexin 3 and Complexin 4 differentially impacts the ON and OFF pathways in mouse retina

Complexins (Cplxs) regulate the speed and Ca₂₊‐sensitivity of synaptic vesicle fusion. It has been shown that all four known Cplxs are present at mouse retinal synapses – at conventional amacrine cell synapses (Cplx 1 to Cplx 3) and at photoreceptor and bipolar cell ribbon synapses (Cplx 3 and Cplx 4) [ K. Reim et al. (2005) J. Cell Biol., 169 , 669‐680]. Electroretinographic recordings in Cplx 3/Cplx 4 double‐knockout (DKO) mice showed perturbed transmission in the outer plexiform layer, and possible changes in the inner plexiform layer [ K. Reim et al. (2009) J. Cell Sci., 122 , 1352–1361]. In the present study, we examined the effects of the absence of Cplx 3 and Cplx 4 on ganglion cell responses. We report that the lack of Cplx 3 and Cplx 4 differentially impacts the ON and OFF pathways. Under photopic conditions, the responses in the cone OFF pathway are largely unaffected, whereas the responses in the cone ON pathway are diminished in Cplx 3/Cplx 4 DKO mice. Under scotopic conditions, both ON and OFF response rates are reduced and high‐sensitivity OFF responses are missing in Cplx 3/Cplx 4 DKO mice. The electrophysiological findings are corroborated by new immunocytochemical findings. We now show that rod spherules contain only Cplx 4. However, both Cplx 3 and Cplx 4 co‐localize in cone pedicles. In the inner plexiform layer, Cplx 3 is present in rod bipolar cell terminals and in amacrine cell processes. Most importantly, Cplx 3 is localized in the lobular appendages of AII amacrine cells, the sites of signal transmission from the primary rod pathway into the OFF pathway in the inner plexiform layer.     

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.     

Localization of neuropeptide Y1 receptor immunoreactivity in the rat retina and the synaptic connectivity of Y1 immunoreactive cells

Neuropeptide Y (NPY), an inhibitory neuropeptide expressed by a moderately dense population of wide-field amacrine cells in the rat retina, acts through multiple (Y1-y6) G-protein-coupled receptors. This study determined the cellular localization of Y1 receptors and the synaptic connectivity of Y1 processes in the inner plexiform layer (IPL) of the rat retina. Specific Y1 immunoreactivity was localized to horizontal cell bodies in the distal inner nuclear layer and their processes in the outer plexiform layer. Immunoreactivity was also prominent in cell processes located in strata 2 and 4, and puncta in strata 4 and 5 of the IPL. Double-label immunohistochemical experiments with calbindin, a horizontal cell marker, confirmed Y1 immunostaining in all horizontal cells. Double-label immunohistochemical experiments, using antibodies to choline acetyltransferase and vesicular acetylcholine transporter to label cholinergic amacrine cell processes, demonstrated that Y1 immunoreactivity in strata 2 and 4 of the IPL was localized to cholinergic amacrine cell processes. Electron microscopic studies of the inner retina showed that Y1-immunostained amacrine cell processes and puncta received synaptic inputs from unlabeled amacrine cell processes (65.2%) and bipolar cell axon terminals (34.8%). Y1-immunoreactive amacrine cell processes most frequently formed synaptic outputs onto unlabeled amacrine cell processes (34.0%) and ganglion cell dendrites (54.1%). NPY immunoreactivity in the rat retina is distributed primarily to strata 1 and 5 of the IPL, and the present findings, thus, suggest that NPY acts in a paracrine manner on Y1 receptors to influence both horizontal and amacrine cells.     

Suppression by zinc of transient OFF responses of carp amacrine cells to red light is mediated by GABA(A) receptors

Modulation by Zn(2+) of ON and OFF responses of transient amacrine cells driven by red- and green-sensitive cones was investigated in isolated, superfused carp retina, using intracellular recording techniques. Zn(2+) selectively abolished the OFF response to red flash of the transient amacrine cells. This Zn(2+) effect was mimicked by GABA application and was blocked by bicuculline, indicating the involvement of GABA(A) receptors. Such differential modulation was observable neither in bipolar cells nor in sustained OFF amacrine cells. It is suggested that the Zn(2+) effect reported here might be due to a direct action of Zn(2+) on GABA(A) receptors of the transient amacrine cells.     

Distribution of CB2 cannabinoid receptor in adult rat retina

Cannabinoid effects are mediated through two receptors, CB1 and CB2. In the retina CB1 has been reported in bipolar cells, gabaergic amacrine cells, horizontal cells, and inner plexiform layer. CB2 receptor mRNA localization was shown in photoreceptors, inner nuclear layer, and ganglion cell layer by using RT‐PCR. The aim of this work was to localize CB2 receptor in the rat retina by using immunocytochemistry. Our results showed that CB2 receptor was localized in retinal pigmentary epithelium, inner photoreceptor segments, horizontal and amacrine cells, cells localized in ganglion cell layer, and in fibers of inner plexiform layer. These results are in agreement with studies using RT‐PCR and provide some additional information about the distribution of CB2 receptor. Further studies are needed to clarify the role of this cannabinoid receptor in the retina. Synapse, 2010. © 2010 Wiley‐Liss, Inc.     

Development of cholinergic amacrine cells is visual activity-dependent in the postnatal mouse retina

In the present study, we used immunocytochemistry to study the temporal and spatial arrangement of mouse cholinergic amacrine cells during postnatal retinal development under normal light/dark cycles and during visual deprivation. Choline acetyltransferase (ChAT)-immunolabeled cells were detected in the neuroblastic layer (NBL) and in the ganglion cell layer (GCL) at postnatal day 0 (P0). Between P3-5, two characteristic cholinergic bands were clearly identified in the inner plexiform layer (IPL). The signal intensity of somas and processes progressively increased over the first 2 postnatal weeks. Around eye opening at P12, cholinergic neurons were mature-like. This early developmental process was not altered by visual deprivation. After eye opening, the space between the two cholinergic bands increased continuously and the spatial regularity index changed constantly, indicating that the cholinergic neurons possibly underwent refinement during later postnatal development. The changes occurring following eye opening were retarded by visual deprivation. The morphologies of photoreceptors, horizontal cells, recoverin-positive OFF-cone bipolar cells, rod bipolar cells, dopaminergic amacrine cells, and Müller cells appeared normal. Their stratification in the outer plexiform layer (OPL) and the IPL was not affected by visual deprivation. However, glial cells grew vertically across the entire thickness of dark-reared retinas. Our results suggest that the development of cholinergic neurons before eye opening is independent of the lighting conditions. Their development after eye opening is greatly impeded by visual deprivation. This visual activity-dependent phase of development may be a critical period for the maturation and synaptic wiring of cholinergic amacrine cells in the mammalian retina.