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Neuropeptide Y (NPY) is a peptide neurotransmitter abundantly expressed in the mammalian retina. Since its discovery, NPY has been studied in retinas of several species, but detailed characterization of morphology, cell-type, and connectivity has never been conducted in larger mammals including humans and pigs. As the pig due to size and cellular composition is a well-suited animal for retinal research, we chose to compare the endogenous NPY system of the human retina to that of pigs to support future research in this field. In the present study, using immunohistochemistry, confocal microscopy and 3D reconstructions, we found NPY to be expressed in GABAergic and calretinin-immunoreactive (-ir) amacrine cells of both species as well as parvalbumin-ir amacrine cells of humans. Furthermore, we identified at least two different types of medium- to wide-field NPY-ir amacrine cells. Finally, we detected likely synaptic appositions between the NPY-ir amacrine cells and melanopsin- and nonmelanopsin-ir ganglion cells, GABAergic and dopaminergic amacrine cells, rod bipolar cells, and horizontal cells, suggesting that NPY-ir cells play diverse roles in modulation of both image and non-image forming retinal signaling. These findings extend existing knowledge on NPY and NPY-expressing cells in the human and porcine retina showing a high degree of comparability. The extensive distribution and connectivity of NPY-ir cells described in the present study further highlights the potential importance of NPY signaling in retinal function.

Intrinsically photosensitive retinal ganglion cells (ipRGCs) mediate the pupillary light reflex, circadian entrainment, and may contribute to luminance and color perception. The diversity of ipRGCs varies from rodents to primates, suggesting differences in their contributions to retinal output. To further understand the variability in their organization and diversity across species, we used immunohistochemical methods to examine ipRGCs in tree shrew (Tupaia belangeri). Tree shrews share membership in the same clade, or evolutionary branch, as rodents and primates. They are highly visual, diurnal animals with a cone-dominated retina and a geniculo-cortical organization resembling that of primates. We identified cells with morphological similarities to M1 and M2 cells described previously in rodents and primates. M1-like cells typically had somas in the ganglion cell layer, with 23% displaced to the inner nuclear layer (INL). However, unlike M1 cells, they had bistratified dendritic fields ramifying in S1 and S5 that collectively tiled space. M2-like cells had dendritic fields restricted to S5 that were smaller and more densely branching. A novel third type of melanopsin immunopositive cell was identified. These cells had somata exclusively in the INL and monostratified dendritic fields restricted to S1 that tiled space. Surprisingly, these cells immunolabeled for tyrosine hydroxylase, a key component in dopamine synthesis. These cells immunolabeled for an RGC marker, not amacrine cell markers, suggesting that they are dopaminergic ipRGCs. We found no evidence for M4 or M5 ipRGCs, described previously in rodents. These results identify some organizational features of the ipRGC system that are canonical versus species-specific.

The present study has taken advantage of publicly available cell type specific mRNA expression databases in order to identify potential genes participating in the development of retinal AII amacrine cells. We profile two such genes, Delta/Notch-like EGF repeat containing (Dner) and nuclear factor I/A (Nfia), that are each heavily expressed in AII amacrine cells in the mature mouse retina, and which conjointly identify this retinal cell population in its entirety when using antibodies to DNER and NFIA. DNER is present on the plasma membrane, while NFIA is confined to the nucleus, consistent with known functions of each of these two proteins. DNER also identifies some other subsets of retinal ganglion and amacrine cell types, along with horizontal cells, while NFIA identifies a subset of bipolar cells as well as Muller glia and astrocytes. During early postnatal development, NFIA labels astrocytes on the day of birth, AII amacrine cells at postnatal (P) day 5, and Muller glia by P10, when horizontal cells also transiently exhibit NFIA immunofluorescence. DNER, by contrast, is present in ganglion and amacrine cells on P1, also labeling the horizontal cells by P10. Developing AII amacrine cells exhibit accumulating DNER labeling at the dendritic stalk, labeling which becomes progressively conspicuous by P10, as it is in maturity. This developmental time course is consistent with a prospective role for each gene in the differentiation of AII amacrine cells.

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.

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. J. Comp. Neurol. 519:433–450, 2011. © 2010 Wiley-Liss, Inc.

The Pde6b(rd10) (rd10) mouse has a moderate rate of photoreceptor degeneration and serves as a valuable model for human autosomal recessive retinitis pigmentosa (RP). We evaluated the progression of neuronal remodeling of second- and third-order retinal cells and their synaptic terminals in retinas from Pde6b(rd10) (rd10) mice at varying stages of degeneration ranging from postnatal day 30 (P30) to postnatal month 9.5 (PNM9.5) using immunolabeling for well-known cell- and synapse-specific markers. Following photoreceptor loss, changes occurred progressively from outer to inner retina. Horizontal cells and rod and cone bipolar cells underwent morphological remodeling that included loss of dendrites, cell body migration, and the sprouting of ectopic processes. Gliosis, characterized by translocation of Müller cell bodies to the outer retina and thickening of their processes, was evident by P30 and became more pronounced as degeneration progressed. Following rod degeneration, continued expression of VGluT1 in the outer retina was associated with survival and expression of synaptic proteins by nearby second-order neurons. Rod bipolar cell terminals showed a progressive reduction in size and ectopic bipolar cell processes extended into the inner nuclear layer and ganglion cell layer by PNM3.5. Putative ectopic conventional synapses, likely arising from amacrine cells, were present in the inner nuclear layer by PNM9.5. Despite these changes, the laminar organization of bipolar and amacrine cells and the ON-OFF organization in the inner plexiform layer was largely preserved. Surviving cone and bipolar cell terminals continued to express the appropriate cell-specific presynaptic proteins needed for synaptic function up to PNM9.5.

Converging evidence indicates that electrical synaptic transmission via gap junctions plays a crucial role in signal processing in the retina. In particular, amacrine and ganglion cells express numerous gap junctions, resulting in extensive electrical networks in the proximal retina. Both connexin36 (Cx36) and connexin45 (Cx45) subunits are widely distributed in the inner plexiform layer (IPL) and therefore are likely contribute to gap junctions formed by a number of ganglion cell subtypes. In the present study, we used the gap junction-permeant tracer Neurobiotin to compare the coupling pattern of different ganglion cell subtypes in wild-type (WT) and Cx36 knockout (KO) mouse retinas. We found that homologous ganglion-to-ganglion cell coupling was lost for two subtypes after deletion of Cx36, whereas two other ganglion cell subtypes retained homologous coupling in the KO mouse. In contrast, deletion of Cx36 resulted in a partial or complete loss of ganglion-to-amacrine cell heterologous coupling in 9 of 10 ganglion cell populations studied. Overall, our results indicate that Cx36 is the predominant subunit of gap junctions in the proximal mouse retina, expressed by most ganglion cell subtypes, and thereby likely plays a major role in the concerted activity generated by electrical synapses.

Each point on the retina is sampled by about 15 types of ganglion cell, each of which is an element in a circuit also containing specific types of bipolar cell and amacrine cell. Only a few of these circuits are well characterized. We found that intracellular injection of Neurobiotin into a specific ganglion cell type targeted by fluorescent markers also stained an asymmetrically branching ganglion cell. It was also tracer-coupled to an unusual type of amacrine cell whose dendrites were strongly asymmetric, coursing in a narrow bundle from the soma in the dorsal direction only. The dendritic field of the ganglion cell stratifies initially in sublamina b (the ON layers), but with few specializations and branches, and then more extensively in sublamina a (the OFF layers) at the level of the processes of the coupled amacrine cell. Intersections of the ganglion and amacrine cell processes contain puncta immunopositive for Cx36. Additionally, we found that the dopaminergic amacrine cell makes contact with both the ganglion cell and the amacrine cell, and that a bipolar cell immunopositive for calbindin synapses onto the sublamina b processes of the ganglion cell. Dopamine D(1) receptor activation reduced tracer flow to the amacrine cells. We have thus targeted and characterized two poorly understood retinal cell types and placed them with two other cell types in a substantial portion of a new retinal circuit. This unique circuit comprised of pronounced asymmetries in the ganglion cell and amacrine cell dendritic fields may result in a substantial orientation bias.

It is now clear that electrical coupling via gap junctions isprevalent across the retina, expressed by each of the fivemain neuronal types. With the introduction of mutants inwhich selective gap junction connexins are deleted, themouse has recently become an important model for studyingthe function of coupling between retinal neurons. In thisstudy we examined the tracer-coupling pattern of ganglioncells by injecting them with the gap junction-permanenttracer Neurobiotin to provide, for the first time, a comprehensivesurvey of ganglion cell coupling in the wildtypemouse retina. Murine ganglion cells were differentiated into22 morphologically distinct subtypes based on somadendriticparameters. Most (16/22) ganglion cell subtypes were tracer-coupled to neighboring ganglion and/or amacrinecells. The amacrine cells coupled to ganglion cellsdisplayed either polyaxonal or wide-field morphologies withextensive arbors. We found that different subtypes of ganglioncells were never coupled to one another, indicatingthat they subserved independent electrical networks. Finally, we found that the tracer-coupling patterns of the 22ganglion cell populations were largely stereotypic acrossthe 71 retinas studied. Our results indicate that electricalcoupling is extensive in the inner retina of the mouse, suggestingthat gap junctions play essential roles in visual information processing.

Members of the Meis subfamily of homeodomain-containing transcription factors play important roles during development and disease. Here we report that the Meis family protein Meis2 is expressed by a subpopulation of gamma-aminobutyric acid (GABA)ergic amacrine (AM) cells in the adult and embryonic retina of different vertebrate species. In mice, Meis2-expressing (Meis2+) AM cells are not cholinergic or dopaminergic, but some are immunoreactive for neuronal nitric oxide synthase (bNOS). About 50% of the mouse Meis2+ AM cell population expresses the calcium-binding protein calretinin, and some Meis2+ AM cells show characteristics of Type II CD-15+ cells. AM cell expression of Meis2 is lost in a conditional knockout mouse model for Pax6, indicating a dependency upon Pax6. Bromodeoxyuridine pulse labeling experiments and immunohistochemical staining for the neuronal marker NeuN in embryonic mouse retinae indicate that Meis2 is an early marker for newly postmitotic AM cells. In addition, taking advantage of the protracted retinal development in humans, we show that newly generated AM cells express Meis2 before adopting the GABAergic or glycinergic neurotransmitter phenotype. As development proceeds, some AM cells lose Meis2 expression concomitantly with the appearance of glycine, while other AM cells retain Meis2 expression after they express GABA. These data identify Meis2 as a suitable marker for the study of AM cell diversity and development in addition to providing evidence for the stepwise specification of the glycinergic and GABAergic neurotransmitter phenotypes during AM cell differentiation.

In the brain and the retina metabotropic glutamate receptors (mGluRs) modulate synaptic transmission; in particular, L-2-amino-4-phosphonobutyrate-sensitive group-III mGluRs are generally presynaptic and provide negative feedback of neurotransmitter release. We performed a comparative immunohistochemical analysis of the distribution of all group-III mGluRs in the mouse retina. mGluR6 expression was limited to the outer plexiform layer. Discrete, punctate immunolabeling, exclusively in the inner plexiform layer (IPL), was observed for each of the remaining group-III mGluRs. mGluR4 immunostaining was most abundant in IPL sublamina 1; mGluR7 immunoreactivity was organized in four bands, corresponding to sublaminae 1– 4; and mGluR8 was localized in two broad bands, one each in the OFF and ON layers of the IPL. mGluR8 immunoreactivity was evident in the OFF plexus of cholinergic amacrine cell processes. Surprisingly, we found little overlap between group-III mGluR immunolabeling and that for the vesicular glutamate transporter VGLUT1. Instead, we found that mGluR4 and mGluR7 were located close to bipolar cell ribbons. No compensatory changes in the distribution of group-III mGluRs, or of several other markers also showing a stratified localization in the IPL, were observed in genetically engineered mice lacking either mGluR4, mGluR8, or both mGluR4 and mGluR8. The unique pattern of expression of each receptor suggests that they have distinct functions in the retina, and their asymmetric distribution in the ON and OFF layers of the IPL suggests distinct roles in the processing of light-ON and light-OFF stimuli. J. Comp. Neurol. 501:931–943, 2007. © 2007 Wiley-Liss, Inc. Indexing terms: immunohistochemistry; mice; knockout; 2-amino-4-phosphonobutyric acid

Studies of retinal ischemia/reperfusion indicate a disparity between the anatomical and functional results; while a large number of rod bipolar cells remain postischemia, there is a significant reduction in the amplitude of the scotopic b-wave of the electroretinogram (ERG). We investigated the alterations in photoreceptor-bipolar cell signaling following ischemia/reperfusion and suggest a mechanism for the decrease in b-wave amplitude. A cation channel probe (agmatine, 1-amino-4-guanidobutane, AGB) was used to assess cellular ion channel activity in neurochemically identified cells secondary to endogenous glutamate release or pharmacological manipulations. By applying the "neurochemical truth point" principle (Sun et al. [2007a] J Comp Neurol, this issue), we have been able to confirm the loss of specific subpopulations of neurons. ERG was used to assess gross retinal function, with parameters of the ERG model providing insight into changes in the phototransduction cascade and sensitivity of postreceptoral glutamate receptors. Following ischemia/reperfusion, rod bipolar cells maintained 2-amino-4-phosphonobutyric acid-responsive metabotropic glutamate receptors and displayed no change in sensitivity to flashes of light as assessed by ERG. Therefore, the loss in b-wave amplitude is likely due to alterations in photoreceptoral glutamate release detected as a change in postsynaptic AGB permeation into rod bipolar cells. Bipolar cell to amacrine cell signaling was also altered. The robust AGB entry into cholinergic amacrine cells was virtually absent in retinas that had undergone ischemia/reperfusion but remained in the AII amacrine cells. Such results suggest a loss of glutamate receptors and/or a change in receptor subunit expression in subpopulations of inner retinal neurons. Although many cells retain their characteristic neurochemical labeling following ischemia/reperfusion, caution should be used when assuming cells participate in functional retinal circuits based solely on the persistence of neurochemical labeling.

The aim of this study was to characterize and classify the displaced amacrine cells in the mouse retina. Amacrine cells in the ganglion cell layer were injected with fluorescent dyes in flat-mounted retinas. Dye-filled displaced amacrine cells were classified according to dendritic field size, horizontal and vertical stratification patterns, and general morphology. We identified 10 different morphological types of displaced amacrine cell. Six of the cell types identified here are novel cell types that have not been described previously in the mouse retina, to the best of our knowledge. The displaced amacrine cells included four types of medium-field cells, with dendritic field diameters of 200–500 μm, and six types of wide-field cells, with dendritic fields extending over 500 μm. Narrow-field displaced amacrine cells, with dendritic field diameters smaller than 200 μm, were not encountered. The most frequently labeled displaced amacrine cell type was the starburst amacrine cell. At least three cell types identified here have nondisplaced counterparts in the inner nuclear layer as well. Displaced amacrine cells display a rich variety of stratification and branching patterns, which surely reflect the wide range of their functional roles in the processing of visual signals in the inner retina. J. Comp. Neurol. 505:177–189, 2007. © 2007 Wiley-Liss, Inc.

The mammalian retina is comprised of six major neuronal cell types and is subdivided into more morphological and physiological subtypes. The transcriptional machinery underlying these subtype fate choices is largely unknown. The LIM-homeodomain protein, Isl1, plays an essential role in central nervous system (CNS) differentiation but its relationship to retinal neurogenesis remains unknown. We report here its dynamic spatiotemporal expression in the mouse retina. Among bipolar interneurons, Isl1 expression commences at postnatal day (P)5 and is later restricted to ON-bipolar cells. The intensity of Isl1 expression is found to segregate the pool of ON-bipolar cells into rod and ON-cone bipolar cells with higher expression in rod bipolar cells. As bipolar cell development proceeds from P5–10 the colocalization of Isl1 and the pan-bipolar cell marker Chx10 reveals the organization of ON-center bipolar cell nuclei to the upper portion of the inner nuclear layer. Further, whereas Isl1 is predominantly a ganglion cell marker prior to embryonic day (E)15.5, at E15.5 and later its expression in nonganglion cells expands. We demonstrate that these Isl1-positive, nonganglion cells acquire the expression of amacrine cell markers embryonically, likely representing nascent cholinergic amacrine cells. Taken together, Isl1 is expressed during the maturation of and is later maintained in retinal ganglion cells and subtypes of amacrine and bipolar cells where it may function in the maintenance of these cells into adulthood. J. Comp. Neurol. 503: 182–197, 2007.

A Neurobiotin-injected OFF parasol cell from midperipheral macaque retina was studied by reconstruction of serial ultrathin sections and compared with ON parasol cells studied previously. In most respects, the synaptic inputs to the two subtypes were similar. Only a few of the amacrine cell processes that provided input to the labeled OFF parasol ganglion cell dendrites made or received inputs within the series, and none of these interactions were with the bipolar cells or other amacrine cells presynaptic to the OFF parasol cell. These findings suggest that the direct inhibitory input to OFF parasol cells originates from other areas of the retina. OFF parasol cells were known to receive inputs from two types of diffuse bipolar cells. To identify candidates for the presynaptic amacrine cells, OFF parasol cells were labeled with Lucifer yellow by using a juxtacellular labeling technique, and amacrine cells known to costratify with them were labeled via immunofluorescent methods. Appositions were observed with amacrine cells containing immunoreactive calretinin, parvalbumin, choline acetylatransferase, and G6-Gly, a cholecystokinin precursor. These findings suggest that the inhibitory input to parasol cells conveys information about several different attributes of visual stimuli and, particularly, about their global properties.

We surveyed wide-field amacrine cells in the mouse, using a large series of retinas from a transgenic strain that expresses the green fluorescent protein (GFP) in isolated retinal cells. Wide-field cells were present in surprising diversity and number. They formed groups that could be defined by arbor depth, arbor size, and soma size. By conventional criteria, these populations of cells make up 11 amacrine cell "types." Five additional types have been reported by others in the mouse. Roughly two-thirds of the wide-field amacrine cells are axon-bearing cells, which have separate dendritic and axonal arbors. The axonal arbor of a single cell sometimes covers the majority of the retinal surface. The axon-bearing cells appear to be centrifugally conducting neurons similar to those studied electrophysiologically in some other species. Although they are classified as independent morphological types, it seems likely that their physiological functions represent variations on a single organizational plan. These cells are present at every level of the inner plexiform layer, which suggests that they affect most of the mouse retina's final outputs to the brain and, by implication, almost all visual function.

With the advent of transgenic mice, much has been learned about the expression and function of gap junctions. Previously, we reported that retinal ganglion cells in mice lacking the neuronal gap junction protein connexin 36 (Cx36) have nearly normal firing patterns at postnatal day 4 (P4) but many more asynchronous action potentials than wild-type mice at P10 (Torborg et al. [2005] Nat. Neurosci. 8:72–78). With the goal of understanding the origin of this increased activity in Cx36–/– mice, we used a transgenic mouse (Deans et al. [2001] Neuron 31:477–485) to characterize the developmental expression of a Cx36 reporter in the retina. We found that Cx36 was first detected weakly at P2 and gradually increased in expression until it reached an adult pattern at P14. Although the onset of expression varied by cell type, we identified Cx36 in the glycinergic AII amacrine cell, glutamatergic cone bipolar cell, and retinal ganglion cells (RGCs). In addition, we used calcium imaging and multielectrode array recording to characterize further the firing patterns in Cx36–/– mice. Both correlated and asynchronous action potentials in P10 Cx36–/– RGCs were significantly inhibited by bath application of an ionotropic glutamate receptor antagonist, indicating that the increase in activity was synaptically mediated. Hence, both the expression patterns and the physiology suggest an increasing role for Cx36-containing gap junctions in suppressing RGC firing between waves during postnatal retinal development. J. Comp. Neurol. 493:309–320, 2005. © 2005 Wiley-Liss, Inc.

Although many effects of gamma-aminobutyric acid (GABA) on retinal function have been attributed to GABA(A) and GABA(C) receptors, specific retinal functions have also been shown to be mediated by GABA(B) receptors, including facilitation of light-evoked acetylcholine release from the rabbit retina (Neal and Cunningham [1995] J. Physiol. 482:363-372). To explain the results of a rich set of experiments, Neal and Cunningham proposed a model for this facilitation. In this model, GABA(B) receptor-mediated inhibition of glycinergic cells would reduce their own inhibition of cholinergic cells. In turn, muscarinic input from the latter to the glycinergic cells would complete a negative-feedback circuitry. In this study, we have used immunohistochemical techniques to test elements of this model. We report that glycinergic amacrine cells are GABA(B) receptor negative. In contrast, our data reveal the localization of GABA(B) receptors on cholinergic/GABAergic starburst amacrine cells. High-resolution localization of GABA(B) receptors on starburst amacrine cells shows that they are discretely localized to a limited population of its varicosities, the majority of likely synaptic-release sites being devoid of detectable levels of GABA(B) receptors. Finally, we identify a glycinergic cell that is a potential muscarinic receptor-bearing target of GABA(B)-modulated acetylcholine release. This target is the DAPI-3 cell. We propose, based on these data, a modification of the Neal and Cunningham model in which GABA(B) receptors are on starburst, not glycinergic amacrine cells.

NADPH diaphorase (NADPHd) histochemistry labels two types of nitrergic amacrine cells in the rabbit retina. Both the large ND1 cells and the small ND2 cells stratify in the middle of the inner plexiform layer, and their overlapping processes produce a dense plexus, which makes it difficult to trace the morphology of single cells. The complete morphology of the ND1 amacrine cells has been revealed by injecting Neurobiotin into large round somata in the inner nuclear layer, which resulted in the labelling of amacrine cells whose proximal morphology and stratification matched those of the ND1 cells stained by NADPHd histochemistry. The Neurobiotin-injected ND1 cells showed strong homologous tracer coupling to surrounding ND1 cells, and double-labelling experiments confirmed that these coupled cells showed NADPHd reactivity. The ND1 amacrine cells branch in stratum 3 of the inner plexiform layer, where they produce a sparsely branched dendritic tree of 400-600 mum diameter in ventral peripheral retina. In addition, each cell gives rise to several fine beaded processes, which arise either from a side branch of the dendritic tree or from the tapering of a distal dendrite. These axon-like processes branch successively within the vicinity of the dendritic field before extending, with little or no further branching, for 3-5 mm from the soma in ventral peripheral retina. Consequently, these cells may span one-third of the visual field of each eye, and their spatial extent appears to be greater than that of most other types of axon-bearing amacrine cells injected with Neurobiotin in this study. The morphology and tracer-coupling pattern of the ND1 cells are compared with those of confirmed type 1 catecholaminergic cells, a presumptive type 2 catecholaminergic cell, the type 1 polyaxonal. cells, the long-range amacrine cells, a novel type of axon-bearing cell that also branches in stratum 3, and a type of displaced amacrine cell that may correspond to the type 2 polyaxonal cell. (C) 2004 Wiley-Liss, Inc.

Morphological studies of rabbit retina have identified ganglion cells resembling "alpha" cells but none resembling cat "beta" cells. Four distinct types of class I cell, similar to alpha cells, were identified, each narrowly stratified and differing from the other three principally in the level of dendritic branching. These four levels of dendritic branching flank the two starburst/cholinergic amacrine cell substrata that mark the middle of sublaminae a and b. Compared with the other three, class Ia2 cells are the largest in cell body and dendritic field size, are sometimes homotypically dye coupled, and have slightly broader dendritic stratification. Class Ia2 and the slightly smaller class Ib2 cells form a paramorphic pair. Compared with class I cells, class II cells have smaller dendritic fields; a greater tendency to "tufted" dendritic branching, as shown in the companion paper; branching at one of three levels of the IPL; and similarly narrow stratification. Class IIa and class Ia1 cells branch at the same level, as do class IIb1 and class Ib1 cells. Class IIb2 cells branch slightly nearer the ganglion cell layer than class Ib2 cells and costratify with "blue-ON" cone bipolar cells. The class IIa and IIb2 cells form a paramorphic pair, whereas class IIb1 cells appear to be unpaired. The four types of class I cell probably correspond to ON- and OFF-center brisk-transient, fast-movement and slow-movement cells, whereas the three types of class II cell probably correspond to ON- and OFF-center brisk-sustained and color-coded ON-center X cells.

Disabled-1 (Dab1) is an adapter molecule in a signaling pathway, stimulated by reelin, that controls cell positioning in the developing brain. It localizes to selected neurons in the nervous system, including the retina, and Dab1-like immunoreactivity is present in AII amacrine cells in the mouse retina. This study was conducted to characterize Dab1-labeled cells in the guinea pig retina in detail using immunocytochemistry, quantitative analysis, and electron microscopy. Dab1 immunoreactivity is present in a class of amacrine cell bodies located in the inner nuclear layer adjacent to the inner plexiform layer (IPL). These cells give rise to processes that ramify the entire depth of the IPL. Double-labeling experiments demonstrated that these amacrine cells make contacts with the axon terminals of rod bipolar cells and that their processes make contacts with each other via connexin 36 in sublamina b of the IPL. In addition, all Dab1-labeled amacrine cells showed glycine transporter 1 immunoreactivity, indicating that they are glycinergic. The density of Dab1-labeled AII amacrine cells decreased from about 3,750 cells/mm 2 in the central retina to 1,725 cells/mm 2 in the peripheral retina. Dab1-labeled amacrine cells receive synaptic inputs from the axon terminals of rod bipolar cells in stratum 5 of the IPL. From these morphological features, Dab1labeled cells of the guinea pig retina resemble the AII amacrine cells described in other mammalian species. Thus, the rod pathway of the guinea pig retina follows the general mammalian scheme and Dab1 antisera can be used to identify AII amacrine cells in the mammalian retina. J. Comp. Neurol. 466:240 –250, 2003. © 2003 Wiley-Liss, Inc. Indexing terms: connexin 36; glycine transporter-1; AII amacrine cells; rod bipolar cells; immunocytochemistry

The mouse retina has become an important model in vision research, mainly because of the wide availability of transgenic animals. In order to study cell function and connectivity in the inner retina, antibodies that differentially stain one cell type, or a small number of cell types, are helpful as markers. Here we characterize the CD15 (3[1-3]-fucosyl-N-acetyllactosamine)-positive cells in the mouse retina using immunofluorescence confocal microscopy and reverse-transcription polymerase chain reaction. CD15 immunoreactivity was observed in two distinct types of amacrine cells and, faintly, in some cone bipolar cells. Type I CD15 amacrine cells are GABAergic wide-field cells that stratify in lamina 3 and 4/5 of the inner plexiform layer. Type II CD15 amacrine cells are also GABAergic and costratify with the dopaminergic tyrosine hydroxylase-positive cells in lamina 1 of the inner plexiform layer. The densities of types I and II CD15 amacrine cells in mid-periphery were 258 cells/mm 2 and 274 cells/mm 2 . Double labeling with several other markers for amacrine cell types showed that neither type belongs to another previously identified subpopulation of amacrine cells. Single-cell RT-PCR showed that CD15 amacrine cells coexpress several AMPA receptors — GluR1, GluR2, and GluR4 being the most common combination. J. Comp. Neurol. 465: 361‐371, 2003. © 2003 Wiley-Liss, Inc. Indexing terms: CD15; inner plexiform layer; amacrine cells; AMPA glutamate receptors

The N-methyl-D-aspartate (NMDA) responses of neurons from within the inner rabbit retina were mapped using a channel permeable cation, 1-amino-4-guanidobutane (agmatine, AGB). Serial sections were subsequently probed with immunoglobulins targeting AGB, glutamate, gamma-aminobutyric acid (GABA), and glycine to visualize the NMDA responses of neurochemical subpopulations of neurons. Most inner retinal subpopulations of neurons demonstrated an NMDA concentration-dependent increase in activation. This NMDA-induced activation displayed a distinct pattern, with the most sensitive class to least sensitive class ranking being GC > GABA cAC > GABA/Gly cAC > Gly cAC > GABA dAC (GC, ganglion cells; AC, amacrine cells; c, conventional; d, displaced; Gly, glycine). The variable NMDA response may reflect differences in NMDA receptor subunit disposition or differences in receptor density. In addition to the variable NMDA activation pattern, we found that virtually all ganglion cells (87%) showed NMDA-gated AGB entry, compared with only 58% of amacrine cells. We conclude that a large cohort of amacrine cells do not possess functional NMDA receptors. In addition to most ganglion cells being activated by NMDA, a large subpopulation displayed the highest sensitivity to NMDA application. The functional significance of this finding is that the ganglion cell population will be the first neuronal class to be susceptible to glutamate-induced neurotoxicity mediated through the NMDA receptor. The addition of betaxolol significantly reduced NMDA-mediated AGB entry into most neuronal groups (ganglion cells, GABA, and glycine amacrine cells), with the greatest effect being on ganglion cells. Betaxolol had no significant effect on NMDA-gated entry of AGB on the GABA/Gly amacrine cell population.

Melanopsin is a novel opsin synthesized in a small subset of retinal ganglion cells. Ganglion cells expressing melanopsin are capable of depolarizing in response to light in the absence of rod or cone input and are thus intrinsically light sensitive. Melanopsin ganglion cells convey information regarding general levels of environmental illumination to the suprachiasmatic nucleus, the intergeniculate leaflet, and the pretectum. Typically, retinal ganglion cells communicate information to central visual structures by receiving input from retinal photoreceptors via bipolar and amacrine cells. Because melanopsin ganglion cells do not require synaptic input to generate light-induced signals, these cells need not receive synapses from other neurons in the retina. In this study, we examined the ultrastructure of melanopsin ganglion cells in the mouse retina to determine the type (if any) of synaptic input these cells receive. Melanopsin immunoreaction product was associated primarily with the plasma membrane of (1) perikarya in the ganglion cell layer, (2) dendritic processes in the inner plexiform layer (IPL), and (3) axons in the optic fiber layer. Melanopsin-immunoreactive dendrites in the inner (ON) region of the IPL were postsynaptic to bipolar and amacrine terminals, whereas melanopsin dendrites stratifying in the outer (OFF) region of the IPL received only amacrine terminals. These observations suggested that rod and/or cone signals may be capable of modifying the intrinsic light response in melanopsin-expressing retinal ganglion cells.

In macaque monkeys the foveal depression forms between fetal day (Fd) 105 and birth (Fd 172 of gestation). Before this, the incipient fovea is identified by a photoreceptor layer comprising cones almost exclusively, a multilayered ganglion cell layer (GCL), and a "domed" profile. Vessels are absent from the central retina until late in development, leading to the suggestion that the GCL in the incipient fovea may be transitorily hypoxic. Vascular endothelial growth factor (VEGF), expressed by both glial and neuronal cells and mediated by the hypoxia-inducible transcription factor (HIF)-1, is the principal factor involved in blood vessel growth in the retina. We examined VEGF expression in macaque retinas between Fd 85 and 4 months postnatal. Digoxygenin-labeled riboprobes were generated from a partial-length human cDNA polymerase chain reaction fragment, detected using fluorescence confocal microscopy, and quantified using Scion Image. High levels of VEGF mRNA were detected in astrocytes associated with developing vessels. We also detected strong expression of VEGF mRNA in the GCL at the incipient fovea prior to Fd 105, with peak labeling in the incipient fovea that declined with distance in nasal and temporal directions. By Fd 152 peak labeling was in two bands associated with development of the inner nuclear layer (INL) capillary plexus: in the inner INL where Müller and amacrine cell somas are located, and in the outer INL where horizontal cells are found. The findings suggest that at the incipient fovea the GCL is hypoxic, supporting the hypothesis that the adaptive significance of the fovea centralis is in ensuring adequate oxygen supply to neuronal elements initially located within the avascular region.

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 Muller cells. The differential labeling suggests the existence of GABA(A) receptor subtypes with different subunit compositions that mediate multiple GABAergic functions in salamander retina.

Starburst amacrine cells in the macaque retina were studied by electron microscopic immunohistochemistry. We found that these amacrine cells make a type of synapse not described previously; they are presynaptic to axon terminals of bipolar cells. We also confirmed that starburst amacrine cells are presynaptic to ganglion cell dendrites and amacrine cell processes. In order to determine the functions of these synapses, we localized acetylcholine receptors using a monoclonal antibody (mAb210) that recognizes human alpha3- and alpha5-containing nicotinic receptors and also antisera against the five known subtypes of muscarinic receptors. The majority of the mAb210-immunoreactive perikarya were amacrine cells and ganglion cells, but a subpopulation of bipolar cells was also labeled. A subset of bipolar cells and a subset of horizontal cells were labeled with antibodies to M3 muscarinic receptors. A subset of amacrine cells, including those that contain cholecystokinin, were labeled with antibodies to M2 receptors. Taken together, these results suggest that acetylcholine can modulate the activity of retinal ganglion cells by multiple pathways.

A fundamental organizing feature of the retina is the presence of regularly spaced distributions of neurons, yet we have little knowledge of how this patterning emerges during development. Among these retinal mosaics, the spatial organization of the dopaminergic amacrine cells is unique: using nearest-neighbor and Vornoi domain analysis, we found that the dopaminergic amacrine cells were neither randomly distributed, nor did they achieve the regularity documented for other retinal cell types. Autocorrelation analysis revealed the presence of an exclusion zone surrounding individual dopaminergic amacrine cells and modeling studies confirmed this organization, as the mosaic could be simulated by a minimal distance spacing rule defined by a broad set of parameters. Experimental studies determined the relative contributions of tangential dispersion, fate determination, and cell death in the establishment of this exclusion zone. Clonal boundary analysis and simulations of proximitydriven movement discount tangential dispersion, while data from bcl-2 overexpressing mice rule out feedback-inhibitory fate-deterministic accounts. Cell death, by contrast, appears to eliminate dopaminergic amacrine cells that are within close proximity, thereby establishing the exclusion zone surrounding individual cells and in turn creating their mosaic regularity. J. Comp. Neurol. 461:123‐136, 2003. © 2003 Wiley-Liss, Inc. Indexing terms: nearest neighbor; Voronoi domain; autocorrelation; density recovery profile; tangential dispersion; fate determination; cell death; bcl-2

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.

We have examined amacrine cells that are calretinin-immunoreactive (-IR) in the macaque monkey retina with the aim of classifying them into morphological and functional subtypes. There are calretinin-IR cells in the fovea and throughout the retina. Their highest density is reached at 1.0 mm from the foveal pit (10,500 cells/mm2) and falls to 2,600/mm2 by 10 mm of eccentricity. Nearest-neighbor statistics for the calretinin-IR cell body distribution indicate a nonregular pattern, with a regularity index of 1.4–1.6. There is an increase or “bump” of cell density 3.5–4.0 mm from the foveal pit, corresponding to the rod photoreceptor density peak. Based on morphological differences, there appear to be three types of amacrine cell that are calretinin-IR. To determine the types, we doubly immunolabeled retinas, from fovea to periphery, for calretinin-IR in combination with other calcium binding proteins and inhibitory amino acid neurotransmitters. Labeling with parvalbumin and calretinin antibodies indicated that 70% of the amacrine cells were solely calretinin-IR, and 30% contained parvalbumin-IR as well. In the same way, 70% of the calretinin-IR amacrine cells colocalized calbindin, but 30% were only calretinin-IR. Among the calretinin/calbindin-colocalized cells, there were small-field and wide-field types. Double labeling with antibodies to calretinin and γ-aminobutyric acid (GABA) and to calretinin and glycine revealed the majority to be glycine-IR, but some were GABA-IR. The glycine-IR population consists mainly of AII amacrine cell types, but clearly another non-AII type is involved. The non-AII glycine-IR population resembles a small- to medium-field diffuse type. The calretinin-IR wide-field type is GABAergic and corresponds to an A19 type. The central, rod-free, fovea contains the calretinin-IR, non-AII glycine-IR type and the calretinin-IR, GABAergic type only. To learn more concerning the circuitry of the calretinin/glycine-IR, non-AII amacrine cell type in isolation from AII amacrine cells, we concentrated on the rod-free fovea, where AII amacrine cells are absent. We performed a serial section electron microscopy (EM) study on four calretinin-IR cells. They were involved with cone pathway circuitry. They got input from ON and OFF midget bipolar cells, reciprocated synapses to these bipolar cells, and provided synapses to ON-center ganglion cells. Thus we have obtained new information on a cone pathway amacrine cell of the central monkey fovea that is involved in the midget system. National Institute of Health; Grant number EY03323 (H.K.); Research to Prevent Blindness grant (Department of Ophthalmology, University of Utah School of Medicine).

Aquaporin 1 (AQP1; also known as CHIP, a channel-forming integral membrane protein of 28 kDa) is the first protein to be shown to function as a water channel and has been recently shown to be present in the rat retina. We previously showed (Kim et al. [1998] Neurosci Lett 244:52-54) that AQP1-like immunoreactivity is present in a certain population of amacrine cells in the rat retina. This study was conducted to characterize these cells in more detail, With immunocytochemistry using specific antisera against AQP1, whole-mount preparations and 50-mum-thick vibratome sections were examined by light and electron microscopy. These cells were a class of amacrine cells, which had symmetric bistratified dendritic trees ramified in stratum 2 and in the border of strata 3 and 4 of the inner plexiform layer (IPL). Their dendritic field diameters ranged from 90 to 230 mum. Double labeling with antisera against AQP1 and gamma-aminobutyric acid or glycine demonstrated that these AQP1-like-immunoreactive amacrine cells were immunoreactive for glycine. Their most frequent synaptic input was from other amacrine cell processes in both sublaminae a and b of the IPL, followed by a few cone bipolar cells. Their primary targets were other amacrine cells and ganglion cells in both sublaminae a and b of the IPL. In addition, synaptic output Onto bipolar cells was rarely observed in sublamina b of the IPL. Thus, the AQP1 antibody labels a class of glycinergic amacrine cells with small to medium-sized dendritic fields in the rat retina. (C) 2002 Wiley-Liss, Inc.

In the mammalian retina, maximum sensitivity is achieved in the rod pathway, which serves dark-adapted vision. Rod bipolar cells carry the highly convergent rod input and make ribbon synapses with two postsynaptic elements in the inner retina. One postsynaptic neuron is the AII amacrine cell, which feeds the rod signal into the cone pathways. The other postsynaptic element is either an S1 or S2 amacrine cell. These two wide-field GABA amacrine cells both make reciprocal synapses with rod bipolar terminals but their individual roles are unknown. AII and S1/S2 dendrites come in close together and form a dyad opposing the presynaptic ribbon, which is the site of glutamate release. Therefore, two postsynaptic neurons sense the very same neurotransmitter yet serve different functions in the rod pathway. This functional diversity could be derived partly from the expression of different glutamate receptors on each postsynaptic element. In this study, we labeled all pre- and postsynaptic combinations and a signal-averaging method was developed to locate glutamate receptor subunits. In summary, GluR2/3 and GluR4 are expressed by AII amacrine cells but not by S1/S2 amacrine cells. In contrast, the orphan subunit delta1/2 is exclusively located on S1 varicosities but not on AII or S2 amacrine cells. These results confirm the prediction of divergence mediated by different glutamate receptors at the rod bipolar dyad. Each different amacrine cell type appears to express specific glutamate receptors. Finally, the differential expression of glutamate receptors by S1 and S2 may partly explain the need for two wide-field GABA amacrine cells with the same feedback connections to rod bipolar terminals.

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. J. Comp. Neurol. 431:424‐436, 2001. © 2001 Wiley-Liss, Inc. Indexing terms: synaptobrevin; synapse; photoreceptor; bipolar cell; amacrine cell

The neuronal form of nitric oxide synthase (NOS-1) has been localized to several cell types in the retinas of experimental animals; however, localization in the human retina has not been definitive. By using in situ hybridization and immunohistochemistry, we have compared the cellular expression and localization of NOS-1 in the rat and human retinas. In both rat and human retinas, NOS-1 is expressed in the inner segments of photoreceptors, cells in the inner nuclear layer, particularly amacrine cells, and retinal ganglion cells. In human cones, NOS-1 is abundantly present in the outer segments. In the rat, optic nerve transection caused a loss of cells that were positive for NOS-1 in the ganglion cell layer. Although a retinal ganglion cell localization has not been reported consistently in the literature, our data clearly localize NOS-1 to the retinal ganglion cells of the rat and human retinas.

In primate retinas, the dendrites of DB3 diffuse bipolar cells are known to receive inputs from cones. The goal of this study was to describe the synaptic connections of DB3 bipolar cell axons in the inner plexiform layer. DB3 bipolar cells in midperipheral retina were labeled with antibodies to calbindin, and their axons were analyzed in serial, ultrathin sections by electron microscopy. Synapses were found almost exclusively at the axonal varicosities of DB3 axon terminals. There were 2.14 synaptic ribbons per varicosity. There were 33 varicosities per DB3 cell, giving an average of 71 ribbons per axon terminal. Because there were 1.5 postsynaptic ganglion cell dendrites per DB3 axonal varicosity, we estimate that there is at least 1 synapse per varicosity onto a parasol ganglion cell dendrite. There were 3.4 input synapses from amacrine cells per axonal varicosity. Among these were feedback synapses to the DB3 bipolar cell axon varicosities, which were made by 47% of the postsynaptic amacrine cell processes. Some of the feedback synapses could be from amacrine cells immunoreactive for cholecystokinin precursor or choline acetyltransferase, because both types of amacrine cells costratify with parasol cells and are known to be presynaptic to bipolar cells. AII amacrine cells were both presynaptic and postsynaptic to DB3 axons, a finding consistent with the large rod input to parasol ganglion cells reported in physiological experiments. DB3 bipolar cell axons also made frequent contacts with neighboring DB3 axons, and gap junctions were always found at these sites.

Amacrine cells in the vertebrate retina can be grouped according to morphology into distinct types, which are organized into characteristic mosaics. Each type is believed to perform a unique role in visual signal processing. Neurotransmitters and calcium binding proteins have served as important markers for amacrine cell populations, yet many types remain to be characterized at the molecular level. We have found that a cyclin kinase inhibitor, p57Kip2, is expressed in a restricted group of amacrine cells in the inner nuclear layer (INL) and ganglion cell layer (GCL) of the rodent retina. Whole-mount antibody staining revealed that the p57Kip2 amacrine cells are evenly distributed across the retina with a density of 1654 +/- 63 cells/mm(2) in the INL and 994 +/- 26 cells/mm(2) in the GCL. These amacrine cells accumulate the major inhibitory neurotransmitter gamma-aminobutyric acid (GABA) but do not accumulate high levels of glycine. In addition, p57Kip2 immunoreactivity does not colocalize with any of the previously identified amacrine cell markers including calbindin, calretinin, parvalbumin, choline acetyltransferase, and tyrosine hydroxylase. To determine whether the p57Kip2 population of amacrine cells is organized into a regular or a random mosaic, nearest neighbor analysis was performed for both the INL and GCL populations. Results from this analysis demonstrated that the p57Kip2-immunoreactive amacrine cells are randomly organized and therefore they are likely to constitute two or more distinct populations. This new molecular marker will serve as a useful tool for future studies on the development and function of amacrine cells in the vertebrate retina.

Presynaptic gamma-aminobutyrate-immunoreactive (GABA+) profiles were mapped in the cyprinid retina with overlay microscopy: a fusion of electron and optical imaging affording high-contrast ultrastructural and immunocytochemical visualization. GABA+ synapses, deriving primarily from amacrine cells (ACs), compose 92% of conventional synapses and 98% of the input to bipolar cells (BCs) in the inner plexiform layer. GABA+ AC synapses, the sign-inverting elements of signal processing, are deployed in micronetworks and distinctive synaptic source/target topologies. Nested feedback micronetworks are formed by three types of links (BC --> AC, reciprocal BC AC synapses) arranged as nested BC [AC --> AC] loops. Circuits using nested feedback can possess better temporal performance than those using simple reciprocal feedback loops. Concatenated GABA+ micronetworks of AC --> AC and AC --> AC --> AC chains are common and must be key elements for lateral spatial, temporal, and spectral signal processing. Concatenated inhibitions may represent exceptionally stable, low-gain, sign-conserving devices for receptive field construction. Some chain elements are GABA immunonegative (GABA-) and are, thus, likely glycinergic synapses. GABA+ synaptic baskets target the somas of certain GABA+ and GABA- cells, resembling cortical axosomatic synapses. Finally, all myelinated intraretinal profiles are GABA+, suggesting that some efferent systems are sources of GABAergic inhibition in the cyprinid retina and may comprise all axosomatic synapses. These micronetworks are likely the fundamental elements for receptive field shaping in the inner plexiform layer, although few receptive field models incorporate them as functional components. Conversely, simple feedback and feedforward synapses may often be chimeras: the result of an incomplete view of synaptic topology.

Cannabinoids have major effects on central nervous system function. Recent studies indicate that cannabinoid effects on the visual system have a retinal component. Immunocytochemical methods were used to localize cannabinoid CB1 receptor immunoreactivity (CB1R-IR) and an endocannabinoid (anandamide and 2-arachidonylglycerol) degradative enzyme, fatty acid amide hydrolase (FAAH)-IR, in the rat retina. Double labeling with neuron-specific markers permitted identification of cells that were labeled with CB1R-IR and FAAH-IR. CB1R-IR was observed in all cells that were protein kinase C-immunoreactive (rod bipolar cells and a subtype of GABA-amacrine cell) as well as horizontal cells (identified by calbindin-IR). There was also punctate CB1R-IR in the distal one-third of the inner plexiform layer (IPL) that could not be assigned to a cell type. FAAH-IR was most prominent in large ganglion cells, whose dendrites projected to a narrow band in the proximal IPL. Weaker FAAH-IR was observed in the soma of horizontal cells (identified by calbindin-IR); the soma of large, but not small, dopamine amacrine cells (identified by tyrosine hydroxylase-IR); and dendrites of orthotopic- and displaced-starburst amacrine cells (identified by choline acetyltransferase-IR) but in less than 50% of the starburst amacrine cell somata. The extensive distribution of CB1R-IR on horizontal cells and rod bipolar cells indicates a role of endocannabinoids in scotopic vision, whereas the more widespread distribution of FAAH-IR indicates a complex control of endocannabinoid release and degradation in the retina. J. Comp. Neurol. 415:80–90, 1999. © 1999 Wiley-Liss, Inc.

Amacrine cells of the rabbit retina were studied by ‘‘photofilling,’’ a photochemical method in which a fluorescent product is created within an individual cell by focal irradiation of the nucleus; and by Golgi impregnation. The photofilling method is quantitative, allowing an estimate of the frequency of the cells. The Golgi method shows their morphology in better detail. The photofilled sample consisted of 261 cells that were imaged digitally in throughfocus series from a previous study (MacNeil and Masland [1998] Neuron 20:971‐982). The Golgi material consisted of 49 retinas that were stained as wholemounts. Eleven of these subsequently were cut in vertical section. Of the many hundreds of cells stained, digital through-focus series were recorded for 208 of the Golgi-impregnated cells. The two methods were found to confirm one another: Most cells revealed by photofilling were recognized easily by Golgi staining, and vice versa. The greater resolution of the Golgi method allowed a more precise description of the cells and several types of amacrine cell were redefined. Two new types were identified. The two methods, taken together, provide an essentially complete accounting of the populations of amacrine cells present in the rabbit retina. Many of them correspond to amacrine cells that have been described in other mammalian species, and these homologies are reviewed. J. Comp. Neurol. 413:305‐326, 1999. r 1999 Wiley-Liss, Inc.

We have used calretinin antibodies to label selectively the mosaic of AII amacrine cells in the macaque retina. Confocal analysis of double-labeled material indicated that AII dendrites spiral down around descending rod bipolar axons before enveloping the synaptic terminals. Processes from a previously observed dopaminergic plexus in the inner nuclear layer were observed to contact the somata of calretinin-positive AII somata. Intracellular neurobiotin injection revealed that AII amacrine cells are tracer coupled to other AII amacrine cells and to some unidentified cone bipolar cells. An analysis of the retinal distribution of macaque AII amacrine cells, including an area in and around the fovea, showed a peak density of approximately 5,000 cells/mm(2) at an eccentricity of 1.5 mm. Staining of AII amacrine cells in central retina with antibodies to calretinin was confirmed by confocal microscopy. These results indicate that calretinin antibodies can be used to label the AII amacrine cell population selectively and that primate AII amacrine cells share many of the features of previously described mammalian AII amacrine cells. The peak AII cell density closely matched the peak sampling rate of scotopic visual acuity. Calculations suggest that, in central macaque retina, where midget ganglion cells are more numerous, AII amacrine cells form the limit of scotopic visual acuity (Wassle et al. [1995] J. Comp. Neurol. 361:537-551). As the ganglion cell density falls rapidly away from the fovea, there is a cross-over point at around 15 degrees eccentricity that matches the inflection point in a psychophysically derived plot of scotopic visual acuity versus eccentricity (Lennie and Fairchild [1994] Vision Res. 34:477-482). The correspondence between the anatomic and psychophysical data supports our interpretation that the anatomic sampling rate of AII amacrine cells limits central scotopic acuity.

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.

The chick retina has three types of cholinergic amacrine cells. We have found that Types I and II differentiate from a common population of postmitotic cells temporarily located in the inner plexiform layer (IPL cells). Golgi staining and immunocytochemistry for choline acetyltransferase (ChAT) and gamma-aminobutyric acid (GABA) were used to trace the development and fate of IPL cells. Transformation of the shape of IPL cells into those typical of both conventional amacrine cells and those displaced to the ganglion cell layer are seen. All IPL cells are doubly immunoreactive, for ChAT and GABA, from the time they appear as a cell population within the inner plexiform layer (IPL) until their separation into the two amacrine cell populations. Polarization and early stages of shape differentiation of both types occur while they are in the IPL, starting in the dorsocentral area in the temporal retina and spreading to the rest of the retina. Three spatial gradients of differentiation are observed: from central-to-peripheral, dorsal-to-ventral, and temporal-to-nasal retina. Our findings suggest that the fate of both types of cells in the chick is determined locally, whereas their postmitotic precursors are within the IPL. The presence of GABA and acetylcholine in both types of amacrine cells at early stages of their morphogenesis, well before they have synaptic interactions, suggests a morphogenetic role for these molecules in inner retinal differentiation.

Nitric oxide is a nonconventional neurotransmitter that is produced as needed by the enzyme nitric oxide synthase (NOS). NOS has been detected in numerous neural structures, including distinct populations of retinal neurons in a variety of vertebrate species. The purpose of this study was to identify NOS-containing cells in the retina and extraretinal ocular tissues of hatched chicks. NOS was detected in frozen sections by using nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry and antisera to neuronal NOS. In the retina, NADPH-diaphorase and NOS immunolabelling were present in four subtypes of amacrine cells, some ganglion cells, efferent fibers, efferent target cells, and neuronal processes in both plexiform layers, whereas diaphorase alone was detected in photoreceptor ellipsoids and Muller cells. In addition, NADPH-diaphorase and immunoreactive NOS were detected in axon bundles and innervation to vascular smooth muscle in the choroid, whereas stromal and endothelial cells in the choroid, scleral chondrocytes, and the retinal pigmented epithelium contained only NADPH-diaphorase. The excitotoxin quisqualate destroyed all but one subtype of NOS-immunoreactive amacrine cell and caused increased NADPH-diaphorase activity in Muller cells. We conclude that nitric oxide is produced by many different cells in the chick eye, including retinal amacrine and ganglion cells, Muller cells, retinal pigmented epithelium, and cells in the choroid, and likely has a broad range of visual and regulatory functions.

Physiological studies of neurons of the inner retina, e.g., of amacrine cells, are now possible in a mammalian retinal slice preparation. The present anatomical study characterizes glycinergic amacrine cells of the rat retina and thus lays the ground for such future physiological and pharmacological experiments. Rat retinae were immunolabeled with antibodies against glycine and the glycine transporter-1 (GLYT-1), respectively. Glycine immunoreactivity was found in approximately 50% of the amacrine and 25% of the bipolar cells. GLYT-1 immunoreactivity was restricted to glycinergic amacrine cells. They were morphologically characterized by the intracellular injection of Lucifer Yellow followed by GLYT-1 immunolabeling. Eight different types of glycinergic amacrine cells could be distinguished. They were all small-field amacrine cells with bushy dendritic trees terminating at different levels within the inner plexiform layer. The well-known AII amacrine cell was encountered most frequently. From our measurements of the dendritic field sizes and the density of glycinergic cells, we estimate that there are enough glycinergic amacrine cells available to make sure that all eight types and possibly more tile the retina regularly with their dendritic fields.

Nerve growth factor is known to stimulate neurite outgrowth and support neuronal survival during embryonic development. We have studied the expression of the nerve growth factor receptor, TrkA, at both mRNA and protein levels during the course of chicken retinal development. Furthermore, we have compared the expression of trkA mRNA with that of the 75-kD low-affinity neurotrophin receptor (p75NTR). RNase protection assay identified peak-levels of trkA mRNA in the late embryonic retina. Using in situ hybridization and immunohistochemistry, we found cells expressing TrkA in both the internal and the external part of the inner nuclear layer, corresponding to amacrine and horizontal cells, respectively. The TrkA-expressing amacrine cell has a unistratified dendritic arborization in the second sublamina of the inner plexiform layer, and may represent the stellate amacrine cell described by Cajal. The horizontal cells, possessing arciform dendrite processes in the outer plexiform layer, showed strong TrkA immunoreactivity in both dendrites and cell bodies. During the course of retinal development, the TrkA-expressing amacrine cells decreased in number, whereas the TrkA-expressing horizontal cells persisted. Because nerve growth factor was expressed where the horizontal cells, but not where the amacrine cells were located, these findings raise the question of whether nerve growth factor could locally support the survival of TrkA-expressing interneurons during retinal development.

The distribution of Connexin43 (Cx43) was examined by immunoblotting and immunofluorescence microscopy in the retinas of five different vertebrates by using a C-terminal specific peptide antibody. The specificity of the antibody was proved on immunoblots, in which it showed cross reactivity with a 43-kDa protein in rat heart homogenates as well as in homogenates of rabbit, rat, chicken, turtle, and fish (carp and zebrafish) retinas. Immunofluorescence histochemistry with retinal cryosections revealed the presence of Cx43 in the retinal pigment epithelium cells of all tested species and in blood vessels of vascularized retinas (fish and rat). Cx43 immunoreactivity was further localized in the stria medullaris of rabbit retina, in the nerve fiber layer of rat retina, most likely in astrocytes, and in the area of the outer limiting membrane of the fish retina, most likely representing Cx43 in Müller glia cells. A punctate Cx43-immunoreactive pattern consistent with gap junctions was also detected in the outer plexiform layer of carp and zebrafish retinas, and a specific amacrine cell type, which ramified in two layers of the inner plexiform layer, was labeled in the zebrafish retina. The present results are in accordance with previous findings showing the abundance of Cx43 in astrocytes, endothelium, and epithelial cells. However, the presence of Cx43 immunoreactivity in a specific population of amacrine cells of the zebrafish retina might indicate that a Cx43-like protein is also expressed in neurons.

The distributions of Shaker subfamily Kv1.1 and Kv1.2 and Shab subfamily Kv2.1 subunits of voltage-gated K+ channels were determined in the retina and ON bipolar cells of goldfish by using double-label light and electron microscopic immunocytochemistry. All labeling to be described was blocked by preabsorption of the primary antibodies with antigen. The retina was labeled throughout with all three antibodies. However, labeling was densest in the inner plexiform layer for Kv1.1, more concentrated in the outer nuclear layer for Kv2.1, and uniform throughout for Kv1.2. All ON mixed rod/cone (mb) and cone (cb) bipolar somata and the proximal portions of their axons and dendrites were labeled for anti-Kv1.1, Kv1.2, and Kv2.1. Labeling of axons rarely extended over the mb axon terminal. Only Kv1.2 antibodies labeled mb bipolar cell dendrites in the outer plexiform layer. No evidence for Kv1.1, 1.2, or 2.1 antibody labeling of OFF bipolar cells was found. Ultrastructurally, Kv1.2-immunoreactivity was associated with the plasma membrane of bipolar cell bodies and with dendrites that make narrow-cleft junctions with cone terminals (ON-type). Kv immunoreactivity was not found associated with presynaptic membranes in the inner plexiform layer and was found only rarely with membranes, postsynaptic to an amacrine cell process. Although both Shaker and Shab subfamilies include delayed rectifiers, their activation properties differ, suggesting differential modulation of K+ conductances in bipolar cells based not only on the presence or absence of rod photoreceptor input but also whether the bipolar cells are of the ON or OFF type.

γ-Aminobutyric acid (GABA) is considered to be a major inhibitory neurotransmitter in the inner plexiform layer of the retinas of all vertebrate species. It is contained in and released from nearly 40% of the amacrine cells and is known to play a major role in many aspects of visual processing. By using well-characterized antibodies to several subunits of the GABAA receptor, we have analyzed their localization on the cell bodies and dendritic trees of two amacrine cell populations in the rabbit retina, which have been either filled intracellularly with Lucifer yellow or stained immunohistochemically. Both populations are selectively stained by intravitreal injection of the fluorescent nuclear dye 4′,6-diaminidin-2-phenylindoldihydrochloride (DAPI). We have found that the most significant concentration of the α1 and β2/3 GABAA receptor subunits is localized to the DAPI-3 type amacrine cell. The perikarya of the DAPI-3 cells are found in the proximal inner nuclear layer and send their processes into two sublayers in sublaminae a and b of the inner plexiform layer. These processes abut but do not directly overlap those of the two mirror-symmetric populations of starburst amacrine cells. Because the cell bodies of the DAPI-3 cells are the only ones in the inner nuclear layer that stain strongly for either the α1 or β2/3 subunits, such staining is a diagnostic feature of these cells. Their processes also constitute the most strongly staining ones found within the inner plexiform layer. The dendritic trees of DAPI-3 cells, which range from about 150 μm up to about 300 μm, exhibit recurvate looping processes reminiscent of those described for directionally selective ganglion cells. In contrast to the DAPI-3 cell, we have also shown that the starburst amacrine cells exhibit no immunoreactivity for the α1 GABAA receptor subunit and very little for the β2/3 subunit. Thus, we have shown that the DAPI-3 cells contain the highest concentrations of the α1 and β2/3 GABAA receptor subunits in the rabbit retina. These cells, which costratify near the processes of both the starburst amacrine cells and the ON-OFF directionally selective ganglion cells, thus, are situated both anatomically and by virtue of their receptor content to potentially interact. J. Comp. Neurol. 393:309–319, 1998. © 1998 Wiley-Liss, Inc.

In this study, we assessed the effects of normal ageing on the number, distribution, and somal area of nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd)-positive (NADPHd+) and tyrosine hydroxylase-immunoreactive (TH-IR) amacrine cells in human and rat retina. By using a double-labelling immunohistochemical technique, we have shown that these two enzymes are located in separate amacrine cell populations in the human retina. In normal human retinas from organ donors, we have shown that there was no change in the number, somal area, or retinal distribution of NADPHd+ neurons over an age range of 19–89 years. In contrast, there was a significant decrease (P < 0.05) of 52% in the total number of TH-IR neurons in the group aged 65–89 years compared with the group aged 19–64 years. CA1 and CA2 TH-IR neurons were reduced by 44% and 55%, respectively. In young (3 months) and old (2 years) rats, the number of NADPHd+ neurons did not decrease with ageing, but the number of TH-IR neurons was significantly reduced by 21% (P < 0.05). In a companion study on monkey retina, we have shown that a postmortem delay of 12.5 hours between death and fixation results in a decrease of 33% in the number of both NADPHd+ and TH-IR neurons in the retina compared with the number in retinas fixed immediately after death. The findings of this study on the two subsets of amacrine cells, therefore, are likely to demonstrate the consequences of ageing in the retina and might contribute to visual impairment in the elderly. J. Comp. Neurol. 389:329–347, 1997. © 1997 Wiley-Liss, Inc.