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.     

Distribution and synaptic connectivity of neuropeptide Y-immunoreactive amacrine cells in the rat retina

Neuropeptide Y (NPY) is a potent bioactive peptide that is widely expressed in the nervous system, including the retina. Here we show that specific NPY immunoreactivity was localized to amacrine and displaced amacrine cells in the rat retina. Immunoreactive cells had a regular distribution across the retina and an overall cell density of 280 cells/mm(2) in the inner nuclear layer (INL) and 90 cells/mm(2) in the ganglion cell layer (GCL). In the INL, most immunoreactive cells were characterized by small cell bodies and fine processes that appeared to ramify primarily in stratum 1 of the inner plexiform layer (IPL). A few cells in the INL also ramified in stratum 3 of the IPL. In the GCL, small to medium immunoreactive cells appeared to ramify primarily in stratum 5 of the IPL. A few immunoreactive processes, originating from somata in the INL and processes in the IPL, ramified in the OPL. NPY-immunoreactive cells contained GABA immunoreactivity, and some amacrine cells also contained tyrosine hydroxylase immunoreactivity. NPY-immunostained processes were most frequently presynaptic to nonimmunostained amacrine and ganglion cell processes and postsynaptic to nonimmunostained amacrine cell processes and cone bipolar cell axonal terminals. These findings indicate that NPY immunoreactivity is present in two populations of amacrine cells, one located in the INL and the other in the GCL, and that these cells mainly form synaptic contacts with other amacrine cells. These observations suggest that NPY-immunoreactive cells participate in multiple circuits mediating visual information processing in the inner retina.     

Identification and characterization of an aquaporin 1 immunoreactive amacrine-type cell of the mouse retina

Using immunocytochemistry, a type of amacrine cell that is immunoreactive for aquaporin 1 was identified in the mouse retina. AQP1 immunoreactivity was found in photoreceptor cells of the outer nuclear layer (ONL) and in a distinct type of amacrine cells of the inner nuclear layer (INL). AQP1-immunoreactive (IR) amacrine cell somata were located in the INL and their processes extended through strata 3 and 4 of the inner plexiform layer (IPL) with thin varicosities. The density of the AQP1-IR amacrine cells increased from 100/mm(2) in the peripheral retina to 350/mm(2) in the central retina. The AQP1-IR amacrine cells comprise 0.5% of the total amacrine cells. The AQP1-IR amacrine cell bodies formed a regular mosaic, which suggested that they represent a single type of amacrine cell. Double labeling with AQP1 and glycine, gamma-aminobutyric acid (GABA) or GAD(65) antiserum demonstrated that the AQP1-IR amacrine cells expressed GABA or GAD(65) but not glycine. Their synaptic input was primarily from other amacrine cell processes. They also received synaptic inputs from a few cone bipolar cells. The primary synaptic targets were ganglion cells, followed by other amacrine cells and cone bipolar cells. In addition, gap junctions between an AQP1-IR amacrine process and another amacrine process were rarely observed. In summary, a GABAergic amacrine cell type labeled by an antibody against AQP1 was identified in the mouse retina and was found to play a possible role in transferring a certain type of visual information from other amacrine or a few cone bipolar cells primarily to ganglion cells.     

Morphological analysis of disabled-1-immunoreactive amacrine cells in the guinea pig retina

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, Dab1-labeled 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.     

Connections of two types of flat cone bipolars in the rabbit retina

The synaptic connections of two types of cone bipolar cells in the rabbit retina were studied with the electron microscope after labeling in vitro with 4′,6-diamidino-2-phenylindole (DAPI), intracellular injection with Lucifer Yellow, and photooxidation (Mills and Massey [1992] J. Comp. Neurol. 321:133). Both types of bipolars belong to the flat variety, because they make basal junctions with a group of four to ten neighboring cone pedicles. One cell type has an axonal arborization that occupies strata 1 through 3 of the inner plexiform layer (IPL). At ribbon synaptic junctions, it is presynaptic to ganglion cell dendrites and to reciprocal dendrites belonging to narrow-field bistratified (AII) amacrine cells. In addition, it contacts and is contacted by other amacrine cell processes of unknown origin. The other cell type has an axonal arborization entirely confined to stratum 2 of the IPL; it is pre- or postsynaptic to a pleomorphic population of amacrine cell processes, and, in particular, it receives input from the lobular appendages of AII. Thus, these two bipolar types probably belong to the off-variety because they make basal junctions with cone photoreceptors and send their axon to sublamina α of the IPL, which is occupied by the dendrites of off-ganglion cells. They are also part of the rod pathway because they receive input from AII amacrine cells. © 1996 Wiley-Liss, Inc.     

Synaptic connections of the narrow-field, bistratified rod amacrine cell (AII) in the rabbit retina.

The synaptic connections of the narrow-field, bistratified rod amacrine cell (AII) in the inner plexiform layer (IPL) of the rabbit retina were reconstructed from electron micrographs of continuous series of thin sections. The AII amacrine cell receives a large synaptic input from the axonal endings of rod bipolar cells in the most vitreal region of the IPL (sublamina b, S5) and a smaller input from axonal endings of cone bipolar cells in the scleral region of the IPL (sublamina a, S1-S2). Amacrine input, localized at multiple levels in the IPL, equals the total number of synapses received from bipolar cells. The axonal endings of cone bipolar cells represent the major target for the chemical output of the AII amacrine cell: these synapses are established by the lobular appendages in sublamina a (S1-S2). Ganglion cell dendrites represent only 4% of the output of the AII amacrine and most of them are also postsynaptic to the cone bipolars which receive AII input. The AII amacrine is not presynaptic to other amacrine cells. Finally, the AII amacrine makes gap junctions with the axonal arborizations of cone bipolars that stratify in sublamina b (S3-S4) as well as with other AII amacrine cells in S5. Therefore, in the rabbit retina 1) the rod pathway consists of five neurons arranged in series: rod-->rod bipolar-->AII amacrine-->cone bipolar-->ganglion cell; 2) it seems unlikely that a class of ganglion cells exists that is exclusively devoted to scotopic functions. In ventral, midperipheral retina, about nine rod bipolar cells converge onto a single AII amacrine, but one of them establishes a much higher proportion of synaptic contacts than the rest. Conversely, each rod bipolar cell diverges onto four AII amacrine cells, but one of them receives the largest fraction of synapses. Thus, within the pattern of convergence and divergence suggested by population studies, preferential synaptic pathways are established.     

Light and electron microscopic analysis of aquaporin 1-like-immunoreactive amacrine cells in the rat retina

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-microm-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 microm. 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.     

Synaptic organization of starburst amacrine cells in rabbit retina: analysis of serial thin sections by electron microscopy and graphic reconstruction

The synaptic organization of starburst amacrine cells was studied by electron microscopy of individual or overlapping pairs of Golgi-impregnated cells. Both type a and type b cells were analyzed, the former with normally placed somata and dendritic branching in sublamina a, and the latter with somata displaced to the ganglion cell layer and branching in sublamina b. Starburst amacrine cells were thin-sectioned horizontally, tangential to the retinal surface, and electron micrographs of each section in a series were taken en montage. Cell bodies and dendritic trees were reconstructed graphically from sets of photographic montages representing the serial sections. Synaptic inputs from cone bipolar cells and amacrine cells are distributed sparsely and irregularly all along the dendritic tree. Sites of termination include the synaptic boutons of starburst amacrine cells, which lie at the perimeter of the dendritic tree in the "distal dendritic zone." In central retina, bipolar cell input is associated with very small dendritic spines near the cell body in the "proximal dendritic zone." The proximal dendrites of type a and type b cells generally lie in planes or "strata" of the inner plexiform layer (IPL), near the margins of the IPL. The boutons and varicosities of starburst amacrine cells, distributed int he distal dendritic zone, lie in the "starburst substrata," which occupy a narrow middle region in each of the two sublaminae, a and b, in rabbit retina. As a consequence of differences in stratification, proximal and distal dendritic zones are potentially subject to different types of input. Type b starburst amacrines do not receive inputs from rod bipolar terminals, which lie mainly in the inner marginal zone of the IPL (stratum 5), but type a cells receive some input from the lobular presynaptic appendages of rod amacrine cells in sublamina a, at the border of strata 1 and 2. There is good correspondence between boutons or varicosities and synaptic outputs of starburst amacrine cells, but not all boutons gave ultrastructural evidence of presynaptic junctions. The boutons and varicosities may be both pre- and postsynaptic. They are postsynaptic to cone bipolar cell and amacrine cell terminals, and presynaptic primarily to ganglion cell dendrites. In two pairs of type b starburst amacrine cells with overlapping dendritic fields, close apposition of synaptic boutons was observed, raising the possibility of synaptic contact between them. The density of the Golgi-impregnation and other technical factors prevented definite resolution of this question. No unimpregnated profiles, obviously amacrine in origin, were found postsynaptic to the impregnated starburst boutons.(ABSTRACT TRUNCATED AT 400 WORDS)     

Synaptic organization of cholinergic amacrine cells in the rhesus monkey retina

In the rhesus monkey retina, choline acetyltransferase (ChAT) immunoreactivity has been used to study the localization and synaptic organization of cholinergic neurons by both light and electron microscopy with peroxidase-antiperoxidase immunohistochemistry. ChAT-containing neurons are a type of amacrine cell with 97.5% of their cell bodies localized to the ganglion cell layer and the remainder in the inner nuclear layer. Their processes arborize in a single narrow band in the inner plexiform layer in a plane dividing the outer two-thirds from the inner one-third of this synaptic region. With electron microscopy, ChAT-immunoreactive amacrine cell processes were observed to be primarily postsynaptic to the diffuse invaginating cone bipolar cells and presynaptic to ganglion cells, although they are both post- and presynaptic to immunohistochemically unlabeled amacrine cell profiles and to ChAT-containing amacrine cell processes as well.     

Melanopsin retinal ganglion cells receive bipolar and amacrine cell synapses

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.     

Localization of GABA, glycine, glutamate and tyrosine hydroxylase in the human retina.

A light microscope study using postembedding immunocytochemistry techniques to demonstrate the common neurotransmitter candidates gamma-aminobutyric acid (GABA), glycine, glutamate, and tyrosine hydroxylase for dopamine has been done on human retina. By using an antiserum to GABA, we found GABA-immunoreactivity (GABA-IR) to be primarily in amacrine cells lying in the inner nuclear layer (INL) or displaced to the ganglion cell layer (GCL). A few stained cells in the INL, which are probably interplexiform cells, were observed to project thin processes towards the outer plexiform layer (OPL). There were heavily stained bands of immunoreactivity in strata 1, 3 and 5 of the inner plexiform layer (IPL). An occasional ganglion cell was also GABA-IR. By using an antiserum to glycine, stained cells were observed at all levels of the INL. Most of these were amacrines, but a few bipolar cells were also glycine-IR. Displaced amacrine cells and large-bodied cells, which are probably ganglion cells, stained in the GCL. The bipolar cells that stained appeared to include both diffuse and midget varieties. The AII amacrine cell of the rod pathway was clearly stained in our material but at a lower intensity than two other amacrine cell types tentatively identified as A8 and A3 or A4. Again, there was stratified staining in the IPL, with strata 2 and 4 being most immunoreactive. An antiserum to glutamate revealed that most of the neurons of the vertical pathways in the human retina were glutamate-IR. Rod and cone photoreceptor synaptic endings labeled as did the majority of bipolar and ganglion cells. The rod photoreceptor stained more heavily than the cone photoreceptor in our material. While both midget and diffuse cone bipolar cell types were clearly glutamate-IR, rod bipolars were not noticeably stained. The most strongly staining glutamate-IR processes of the IPL lay in the outer half, in sublamina a. The antiserum to tyrosine hydroxylase (TOH) revealed two different amacrine cell types. Strongly immunoreactive cells (TOH1) had their cell bodies in the INL and their dendrites ramified in a dense plexus in stratum 1 of the IPL. Fine processes arising from their cell bodies or from the stratum 1 plexus passed through the INL to reach the OPL but did not produce long-ranging ramifications therein. The less immunoreactive amacrines (TOH2) lay in the INL, the center of the IPL or the GCL and emitted thick dendrites that were monostratified in stratum 3 of the IPL.     

Development of the rhesus monkey retina: II. A three-dimensional analysis of the sequences of synaptic combinations in the inner plexiform layer

The inner plexiform layer (IPL) of the retina provides a useful model for ultrastructural analysis of synaptic development. In primates, the IPL consists of numerous combinations of neuronal contacts that assume the morphological configuration of either conventional or ribbon synapses. We have determined the sequential development of these combinations by analyzing serial electron microscopic sections from fetal rhesus monkeys. Our analysis reveals an orderly emergence of various pre- and postsynaptic elements: (1) patches of dense filamentous membrane first appear on the dendrites of ganglion (G) cells; (2) membrane densities on ganglion cell dendrites then become apposed to amacrine (A) cell processes still lacking their own membrane densities and synaptic vesicles; (3) amacrine cell processes acquire membrane specializations associated with vesicles at the sites apposing ganglion cell dendrites, thereby establishing the first morphologically complete, A----G subtype of conventional synapse; (4) pairs of amacrine cell processes form A----A subtypes of conventional synapses; (5) next, monad ribbon synapses are established between bipolar (B) and ganglion or amacrine cell processes (B----G; B----A); (6) the three subclasses of dyad ribbon synapses (B----GG; B----GA; B----AA) are subsequently formed by the introduction of additional amacrine or ganglion cell processes in the dyad synapse; (7) concurrently, processes of some amacrine and interplexiform (I) cells form a feedback circuit with bipolar cell axons (A----B; I----B), thereby completing the synaptic microcircuitry of the IPL. Present findings provide evidence that the sequence of synaptic differentiation in the IPL proceeds from the postsynaptic to the presynaptic site. Furthermore, lateral interactions (A----G and A----A) are established prior to the formation of the "straight signal" pathway from photoreceptors (P) via bipolar cells to ganglion cells (P----B----G). Observed developmental events provide new insight into the order of establishment of local neuronal circuits in the primate retina.     

Synaptic analysis of amacrine cells with neuropeptide Y-like immunoreactivity in turtle retina

Neuropeptide Y-like immunoreactivity has been localized previously within three classes of amacrine cells in the turtle retina. We have used the avidin-biotin with horseradish peroxidase technique to label these neurons for examination at the ultrastructural level to answer the following questions. Where are the synaptic contacts of these neurons made? What types of neurons are involved pre- and postsynaptically? What is the intracellular distribution of the immunoreactivity? Processes with neuropeptide Y-like immunoreactivity were located primarily within three regions of the inner plexiform layer: stratum 1, stratum 3, and at the border between strata 4 and 5. In all three regions the processes with neuropeptide Y-like immunoreactivity received synaptic contacts from both unlabeled amacrine and bipolar cells, but the majority of the synaptic input in all three regions was from unlabeled amacrine cells. Processes with neuropeptide Y-like immunoreactivity were presynaptic to unlabeled amacrine cells in all three regions, but also formed contacts onto unlabeled bipolar cells in the region between strata 4 and 5. The immunoreactivity within these cells gave rise to a diffuse reaction product that was distributed throughout the cytoplasm and within large vesicles. This localization of neuropeptide Y-like immunoreactivity within large vesicles suggests that this peptide may play a neuromodulatory role. Such a role would be consistent with previous studies of neuropeptides in the turtle retina.     

Multiple cell types form the VIP amacrine cell population

Amacrine cells are a heterogeneous group of interneurons that form microcircuits with bipolar, amacrine and ganglion cells to process visual information in the inner retina. This study has characterized the morphology, neurochemistry and major cell types of a VIP-ires-Cre amacrine cell population. VIP-tdTomato and -Confetti (Brainbow2.1) mouse lines were generated by crossing a VIP-ires-Cre line with either a Cre-dependent tdTomato or Brainbow2.1 reporter line. Retinal sections and whole-mounts were evaluated by quantitative, immunohistochemical, and intracellular labeling approaches. The majority of tdTomato and Confetti fluorescent cell bodies were in the inner nuclear layer (INL) and a few cell bodies were in the ganglion cell layer (GCL). Fluorescent processes ramified in strata 1, 3, 4, and 5 of the inner plexiform layer (IPL). All tdTomato fluorescent cells expressed syntaxin 1A and GABA-immunoreactivity indicating they were amacrine cells. The average VIP-tdTomato fluorescent cell density in the INL and GCL was 535 and 24 cells/mm², respectively. TdTomato fluorescent cells in the INL and GCL contained VIP-immunoreactivity. The VIP-ires-Cre amacrine cell types were identified in VIP-Brainbow2.1 retinas or by intracellular labeling in VIP-tdTomato retinas. VIP-1 amacrine cells are bistratified, wide-field cells that ramify in strata 1, 4, and 5, VIP-2A and 2B amacrine cells are medium-field cells that mainly ramify in strata 3 and 4, and VIP-3 displaced amacrine cells are medium-field cells that ramify in strata 4 and 5 of the IPL. VIP-ires-Cre amacrine cells form a neuropeptide-expressing cell population with multiple cell types, which are likely to have distinct roles in visual processing.     

Aspartate aminotransferase-like immunoreactivity in the guinea pig and monkey retinas

The excitatory amino acids, aspartate and glutamate, have been proposed as retinal neurotransmitters. Aspartate aminotransferase (AAT) is an enzyme which is involved in the routine metabolism of these amino acids and may be involved in the specific synthesis of glutamate and/or aspartate for use as a neurotransmitter. On the basis of the hypothesis that increased levels of aspartate aminotransferase may reflect a transmitter role for aspartate and/or glutamate, we have localized aspartate aminotransferase in the guinea pig and cynamolgus monkey retinas with light and electron microscopic immunohistochemical techniques. AAT-like immunoreactivity is localized to the cones of guinea pig retina and to monkey rods. Both species contain a subpopulation of immunoreactive amacrine cells as well as a subpopulation of immunoreactive cells in the ganglion cell layer. Immunostaining is seen in bipolar cells and terminals in the monkey but not in the guinea pig retina. We have performed quantitative analysis of the immunoreactive staining in the outer plexiform layer and described the synaptic organization of immunoreactive processes in the inner plexiform layer (IPL). Labeled amacrine processes in both species form synaptic contacts predominantly to and from bipolar terminals in the inner third of the IPL and to and from other amacrine and small unidentified processes in the outer portion of the IPL. The majority of labeled bipolar terminals in the monkey retina are seen in the inner third of the IPL where they synapse exclusively onto amacrine processes. Labeled bipolar terminals in the outer third of the IPL occasionally synapse onto ganglion processes.     

Serotonergic and serotonin-accumulating neurons in the goldfish retina

Autoradiography of goldfish retinas incubated in micromolar levels of 3H-serotonin displayed 3 kinds of labeled somas in the inner nuclear layer: S1 amacrine cells with heavy labeling, large somas, and a sparse distribution (approximately 93/mm2); S2 amacrine cells with moderate labeling, smaller somas, and a denser distribution (approximately 500/mm2); and a subset of bipolar cells with light labeling, small somas, and a very dense distribution (approximately 4000/mm2). Serotonin-like immunoreactivity was observed only in S1 amacrine cells and their synaptic terminals. Radiolabeled terminals in the inner plexiform layer formed 4 strata that were differentially assigned to the 3 cell types. S1 amacrine cells arborized in sublayers 1 and 5, received inputs from type a1 bipolar cells and amacrine cells, and made synapses on other amacrine cells, type a1 bipolar cells and unidentified processes. Thus, S1 amacrine cells seem to receive significant input from "off-center" pathways. S2 amacrine cells arborized in sublayer 3 and made synapses onto amacrine cells. Labeled bipolar cell terminals were exclusively located in sublayer 2 and were identified as type a2 mixed rod-cone bipolar cells. We conclude that the S1 amacrine cell is truly serotonergic and that radiolabeling of S2 amacrine cells and type a2 bipolar cells is due to cross-specificity for another carrier or processes unrelated to their neurochemical identities. These observations partially reconcile many previous observations on the types, numbers, and synaptologies of teleost retinal neurons identified by different markers for indoleaminergic transmission.     

Morphological analysis of the hyperpolarization-activated cyclic nucleotide-gated cation channel 1 (HCN1) immunoreactive bipolar cells in the rabbit retina

Hyperpolarization-activated cation currents (I(h)) have been identified in neurons in the central nervous system, including the retina. There is growing evidence that these currents, mediated by the hyperpolarization-activated cyclic nucleotide-gated cation channel (HCN), may play important roles in visual processing in the retina. This study was conducted to identify and characterize HCN1-immunoreactive (IR) bipolar cells by immunocytochemistry, quantitative analysis, and electron microscopy. The HCN1-IR bipolar cells were a subtype of OFF-type cone bipolar cells and comprised 10% of the total number of cone bipolar cells. The axons of the HCN1-IR cone bipolar cells ramified narrowly in the border of strata 1 and 2 of the inner plexiform layer (IPL). These cells formed a regular distribution, with a density of 1,825 cells/mm(2) at a position 1 mm ventral to the visual streak, falling to 650 cells/mm(2) in the ventral periphery. Double-labeling experiments demonstrated that their axons stratified narrowly within and slightly proximal to the OFF-starburst amacrine cell processes. In the IPL, they were presynaptic to amacrine cell processes. The most frequent postsynaptic dyads formed of HCN1-IR bipolar cell axon terminals are pairs composed of both amacrine cell processes. These results suggest that these HCN1-IR cone bipolar cells might be the same as the DAPI-Ba1 bipolar population, and might therefore be involved in a direction-selective mechanism, providing inputs to the OFF-starburst amacrine cells and/or the OFF-plexus of the ON-OFF ganglion cells.     

Synaptic organization of substance P-like immunoreactive amacrine cells in goldfish retina

A class of amacrine cells in the goldfish retina displays substance P-like immunoreactivity (SPIR). We studied the synaptic organization of SPIR amacrine cells by electron microscopical immunocytochemistry. Amacrine cells showing SPIR have processes which ramify in a very narrow band in layer 3 of the inner plexiform layer. SPIR is restricted to large dense-cored vesicles (DCVs), which are distributed throughout the dendrites. Processes labeled with SPIR contain a mixture of DCVs and numerous small agranular vesicles. Of 88 synaptic contracts analyzed, SPIR processes occurred as the presynaptic element 57 times and as the postsynaptic element 31 times. SPIR processes made synapses upon amacrine and ganglion cell dendrites with equal frequency and received synaptic input from both amacrine and bipolar cells. The stratification of SPIR amacrine cells in proximal sublamina a suggests that their synaptic interactions are restricted to "off" and "on-off" neurons. However, this is in contrast to published electrophysiological data. Possible explanations for this discrepancy are discussed in detail.     

Vasoactive intestinal polypeptide-containing cells in the rabbit retina: immunohistochemical localization and quantitative analysis

Vasoactive intestinal polypeptide (VIP) possesses neuroactive properties in the nervous system. In this study we characterized VIP immunoreactive neurons in the rabbit retina to provide a basis for a better understanding of the role of this peptide in retinal functions and to further define the morphology of wide-field amacrine cells. VIP immunoreactivity was demonstrated in colchicine-treated retinas. Immunolabeling was observed in amacrine cells located in the proximal inner nuclear layer and, occasionally, in the ganglion cell layer and inner plexiform layer (IPL). Varicose fibers were distributed in laminae 1, 3, and 5 of the IPL. The distribution of VIP immunoreactive cells showed a peak of approximately 50 cells/mm2 in the visual streak and minimum values of approximately 12 cells/mm2 in the peripheral retina. The total number of VIP immunopositive neurons was estimated to be about 11,000. Cell body diameters in the visual streak (8-9 microns) were slightly smaller than those measured in the dorsal or in the ventral retina (9-10 microns). The distribution of nearest neighbor distances (approximately 109 microns in the visual streak and approximately 99 microns in the peripheral retina) showed that VIP immunoreactive neurons were nonrandomly spaced. Labeled neurons emitted one to three thick primary processes, arborizing in secondary processes and collaterals rich in varicosities; these processes often crossed among different IPL laminae. Arborization fields of individual cells overlapped extensively. In the dorsal retina, estimated areas of single arborization fields were larger and processes had lower branching frequency than in the visual streak and in the ventral retina. On the whole, VIP immunoreactive amacrine cells gave rise to a loose meshwork of fibers in the IPL. These characteristics indicate VIP is contained in a class of wide-field amacrine cells and is likely to be involved in widespread regulatory or modulatory functions rather than in the direct transmission of visual information through the retina.     

Ultrastructural demonstration ofl-glutamate decarboxylase and cysteinesulfinic acid decarboxylase in rat retina by immunocytochemistry

The γ-aminobutyric acid (GABA) synthesizing enzyme,l-glutamate decarboxylase (GAD), and the taurine synthesizing enzyme, cysteinesulfinic acid decarboxylase (CSAD) have been localized in rat retina at the ultrastructural level by indirect immunoelectron microscopy. GAD immunoreactivity (GAD-IR) was seen only in some amacrine cells and their terminals. CSAD immunoreactivity (CSAD-IR) was found in most retinal neuronal types and their processes including photoreceptor cells (rod and cone cells), bipolar cells, amacrine cells and ganglion cells. The GAD-IR positive amacrine terminals have been found to make synaptic contact with other GAD-IR negative bipolar and amacrine terminals, and ganglion cell dendrites. Most of the GAD-IR positive terminals are presynaptic. Occasionally, GAD-IR positive amacrine terminals are postsynaptic to another amacrine terminal or ganglion cell body. In the inner plexiform layer, CSAD-IR positive amacrine terminals also make synaptic contacts with other nerve terminals, similar to that of GAD-IR positive amacrine terminals. In addition, CSAD-IR positive bipolar terminals make synaptic contact with some CSAD-IR positive as well as negative amacrine terminals. Both CSAD-IR positive amacrine and bipolar terminals are mostly presynaptic to other CSAD-IR negative terminals. In the outer plexiform layer, CSAD-IR was found to be associated with synaptic vesicles and the synaptic membrane in certain cone pedicles and rod spherules. It is concluded that only a fraction of amacrine cells in rat retina may use GABA as a neurotransmitter. The presence of CSAD-IR in some amacrine, bipolar, photoreceptor and ganglion cells in rat retina is compatible with the notion that taurine may play some important roles, such as those of neurotransmitter or neuromodulator in mammalian retina.