Selective synaptic connections in the retinal pathway for night vision

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

Synaptic inputs onto small bistratified (blue‐ON/yellow‐OFF) ganglion cells in marmoset retina

The inner plexiform layer of the retina contains functional subdivisions, which segregate ON and OFF type light responses. Here, we studied quantitatively the ON and OFF synaptic input to small bistratified (blue‐ON/yellow‐OFF) ganglion cells in marmosets (Callithrix jacchus). Small bistratified cells display an extensive inner dendritic tier that receives blue‐ON input from short‐wavelength‐sensitive (S) cones via blue cone bipolar cells. The outer dendritic tier is sparse and is thought to receive yellow‐OFF input from medium (M)‐ and long (L)‐wavelength‐sensitive cones via OFF diffuse bipolar cells. In total, 14 small bistratified cells from different eccentricities were analyzed. The cells were retrogradely labeled from the koniocellular layers of the lateral geniculate nucleus and subsequently photofilled. Retinal preparations were processed with antibodies against the C‐terminal binding protein 2, the AMPA receptor subunit GluR4, and/or gephyrin to identify bipolar and/or amacrine input. The results show that the synaptic input is evenly distributed across the dendritic tree, with a density similar to that reported previously for other ganglion cell types. The population of cells showed a consistent pattern, where bipolar input to the inner tier is about fourfold greater than bipolar input to the outer tier. This structural asymmetry of bipolar input may help to balance the weight of cone signals from the sparse S cone array against inputs from the much denser M/L cone array. J. Comp. Neurol. 517:655–669, 2009. © 2009 Wiley‐Liss, Inc.     

Synaptic input to small bistratified (blue-ON) ganglion cells in the retina of a new world monkey, the marmoset Callithrix jacchus.

Small bistratified (blue-ON) ganglion cells in the primate retina are involved in processing short wavelength sensitive cone signals. These ganglion cells stratify in both the ON- and OFF-sublamina of the inner plexiform layer. We investigated the origin of synaptic input to the small bistratified ganglion cell in the retina of a New World primate, the marmoset Callithrix jacchus. Two small bistratified cells from peripheral retina were intracellularly filled with Lucifer Yellow, subsequently photoconverted and processed for electron microscopy. Serial ultrathin sections were cut through portions of each cell, and these were analysed in the electron microscope. The majority of synaptic input (about 84%) to both the inner and outer tier of dendrites was from amacrine cells. Both dendritic tiers also received bipolar cell input. These findings are consistent with predictions from physiological studies that synaptic input to the inner and outer tier of small bistratified cells should be excitatory. However, the tiny fraction of total input supplied from bipolar cells to the outer tier is not consistent with the strong excitatory OFF response in cells of this pathway.     

Ionotropic glutamate receptors during the development of the chick retina.

Glutamate is the main neurotransmitter of photoreceptors, bipolar cells, and ganglion cells of the vertebrate retina. Three main classes of ionotropic glutamate receptors comprising different subunits can be distinguished: AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxasolepropionate), KA (kainate), and NMDA (N-methyl-D-aspartate). This study was undertaken to characterize the AMPA (GluR1, GluR2/3, and GluR4), KA (GluR5/6/7), and NMDA (NR1) ionotropic glutamate receptor subunits and to determine their distribution during the development of the chick retina by Western blotting and immunohistochemistry. Western blotting analysis at 1 day after hatching indicated that the antibodies against GluR1, 2/3, 4, and 5/6/7 and NR1 recognized specifically a single band of 100-110 kDa. In turn, immunohistochemistry at P1 showed that all subunits were expressed in cells of the inner nuclear and ganglion cell layers of the chick retina, mostly amacrine and ganglion cells, and their processes in the inner plexiform layer. In addition, stained processes in the outer plexiform layer were observed with the antibodies against GluR2/3, GluR4, and GluR5/6/7. Although all subunits appeared around E5-E6 in the prospective ganglion cell layer, and later in the prospective inner nuclear layer, the distribution of cells containing these glutamate receptor subunits revealed distinct ontogenetic patterns. This multiplicity of glutamate receptors may contribute to different processes that occur in the chick retina during development.     

Ectopic retinal ON bipolar cell synapses in the OFF inner plexiform layer: Contacts with dopaminergic amacrine cells and melanopsin ganglion cells

A key principle of retinal organization is that distinct ON and OFF channels are relayed by separate populations of bipolar cells to different sublaminae of the inner plexiform layer (IPL). ON bipolar cell axons have been thought to synapse exclusively in the inner IPL (the ON sublamina) onto dendrites of ON‐type amacrine and ganglion cells. However, M1 melanopsin‐expressing ganglion cells and dopaminergic amacrine (DA) cells apparently violate this dogma. Both are driven by ON bipolar cells, but their dendrites stratify in the outermost IPL, within the OFF sublamina. Here, in the mouse retina, we show that some ON cone bipolar cells make ribbon synapses in the outermost OFF sublayer, where they costratify with and contact the dendrites of M1 and DA cells. Whole‐cell recording and dye filling in retinal slices indicate that type 6 ON cone bipolars provide some of this ectopic ON channel input. Imaging studies in dissociated bipolar cells show that these ectopic ribbon synapses are capable of vesicular release. There is thus an accessory ON sublayer in the outer IPL. J. Comp. Neurol. 517:226‐244, 2009. © 2009 Wiley‐Liss, Inc.     

Morphology and connectivity of the small bistratified A8 amacrine cell in the mouse retina

Amacrine cells comprise ～30 morphological types in the mammalian retina. The synaptic connectivity and function of a few γ‐aminobutyric acid (GABA)ergic wide‐field amacrine cells have recently been studied; however, with the exception of the rod pathway‐specific AII amacrine cell, the connectivity of glycinergic small‐field amacrine cells has not been investigated in the mouse retina. Here, we studied the morphology and connectivity pattern of the small‐field A8 amacrine cell. A8 cells in mouse retina are bistratified with lobular processes in the ON sublamina and arboreal dendrites in the OFF sublamina of the inner plexiform layer. The distinct bistratified morphology was first visible at postnatal day 8, reaching the adult shape at P13, around eye opening. The connectivity of A8 cells to bipolar cells and ganglion cells was studied by double and triple immunolabeling experiments by using various cell markers combined with synaptic markers. Our data suggest that A8 amacrine cells receive glutamatergic input from both OFF and ON cone bipolar cells. Furthermore, A8 cells are coupled to ON cone bipolar cells by gap junctions, and provide inhibitory input via glycine receptor (GlyR) subunit α1 to OFF cone bipolar cells and to ON A‐type ganglion cells. Measurements of spontaneous glycinergic postsynaptic currents and GlyR immunolabeling revealed that A8 cells express GlyRs containing the α2 subunit. The results show that the bistratified A8 cell makes very similar synaptic contacts with cone bipolar cells as the rod pathway‐specific AII amacrine cell. However, unlike AII cells, A8 amacrine cells provide glycinergic input to ON A‐type ganglion cells. J. Comp. Neurol. 523:1529–1547, 2015. © 2015 Wiley Periodicals, Inc.     

GABA-like immunoreactivity in the macaque monkey retina: a light and electron microscopic study

The distribution of GABA-like immunoreactivity in the macaque monkey retina was studied by using postembedding techniques on semithin and ultrathin sections. At the light microscopic level, both inner and outer plexiform layers showed strong GABA-like immunoreactivity in the central retina. All the horizontal cells, some bipolar cells, 30-40% of amacrine cells, occasional interplexiform cells, and practically all displaced amacrine cells were labeled. In the peripheral retina (beyond 5 mm eccentricity), the outer plexiform layer and the horizontal cells were not labeled, but all other cell types showed the same labeling pattern as in the central retina. Synapses of the inner plexiform layer involving a pre- or postsynaptic GABA-labeled process were studied electron microscopically. Synapses involving a GABA-labeled presynaptic amacrine cell process made up 80% of the synapses observed. These GABA-labeled amacrine processes synapsed onto amacrine, bipolar, and ganglion cell processes as well as onto amacrine and ganglion cell bodies. Synapses involving a postsynaptic GABA-labeled process made up 20% of the synapses studied. The GABA-like immunoreactive processes were postsynaptic to bipolar cells at the dyads and to amacrine cells at conventional synapses.     

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

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

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.     

ON cone bipolar cell axonal synapses in the OFF inner plexiform layer of the rabbit retina

Analysis of the rabbit retinal connectome RC1 reveals that the division between the ON and the OFF inner plexiform layer (IPL) is not structurally absolute. ON cone bipolar cells make noncanonical axonal synapses onto specific targets and receive amacrine cell synapses in the nominal OFF layer, creating novel motifs, including inhibitory crossover networks. Automated transmission electron microscopic imaging, molecular tagging, tracing, and rendering of ～400 bipolar cells reveals axonal ribbons in 36% of ON cone bipolar cells, throughout the OFF IPL. The targets include γ‐aminobutyrate (GABA)‐positive amacrine cells (γACs), glycine‐positive amacrine cells (GACs), and ganglion cells. Most ON cone bipolar cell axonal contacts target GACs driven by OFF cone bipolar cells, forming new architectures for generating ON–OFF amacrine cells. Many of these ON–OFF GACs target ON cone bipolar cell axons, ON γACs, and/or ON–OFF ganglion cells, representing widespread mechanisms for OFF to ON crossover inhibition. Other targets include OFF γACs presynaptic to OFF bipolar cells, forming γAC‐mediated crossover motifs. ON cone bipolar cell axonal ribbons drive bistratified ON–OFF ganglion cells in the OFF layer and provide ON drive to polarity‐appropriate targets such as bistratified diving ganglion cells (bsdGCs). The targeting precision of ON cone bipolar cell axonal synapses shows that this drive incidence is necessarily a joint distribution of cone bipolar cell axonal frequency and target cell trajectories through a given volume of the OFF layer. Such joint distribution sampling is likely common when targets are sparser than sources and when sources are coupled, as are ON cone bipolar cells. J. Comp. Neurol. 521:977–1000, 2013. © 2012 Wiley Periodicals, Inc.     

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

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

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.     

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

The gamma-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.     

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.     

Characterization of nitric oxide signaling pathways in the mouse retina

Nitric oxide (NO) is a gaseous neuromodulator with physiological functions in every retinal cell type. NO is synthesized by several nitric oxide synthases (NOS) and often functions through its second messenger, cyclic guanosine monophosphate (cGMP), and protein kinase G (PKG). This study combined NO imaging, immunocytochemistry, biochemistry, and molecular biology to localize NO and its downstream signaling pathways in the mouse retina. Neuronal NOS (nNOS) was localized primarily in puncta in the inner plexiform layer, in amacrine cells, and in somata in the ganglion cell layer. Endothelial NOS was in blood vessels. Light‐stimulated NO production imaged with diaminofluorescein was present in somata in the inner nuclear layer and in synaptic boutons in the inner plexiform layer. The downstream target of NO, soluble guanylate cyclase (sGC), was in somata in the inner and outer nuclear layers and in both plexiform layers. Cyclic GMP immunocytochemistry was used functionally to localize sGC that was activated by an NO donor in amacrine, bipolar, and ganglion cells. Cyclic GMP‐dependent protein kinase (PKG) Iα was found in bipolar cells, ganglion cells, and both plexiform layers, whereas PKG II was found in the outer plexiform layer, amacrine cells, and somata in the ganglion cell layer. This study shows that the NO/cGMP/PKG signaling pathway is functional and widely distributed in specific cell types in the outer and inner mouse retina. A better understanding of these signaling pathways in normal retina will provide a firm basis for targeting their roles in retinal pathology. J. Comp. Neurol. 520:4204–4217, 2012. © 2012 Wiley Periodicals, Inc.     

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

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

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.     

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

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

Microcircuitry of bipolar cells in cat retina

We have studied 15 bipolar neurons from a small patch (14 X 120 micron) of adult cat retina located within the area centralis. From electron micrographs of 189 serial ultrathin sections, the axon of each bipolar cell was substantially reconstructed with its synaptic inputs and outputs by means of a computer-controlled reconstruction system. Based on differences in stratification, cytology, and synaptic connections, we identified eight different cell types among the group of 15 neurons: one type of rod bipolar and seven types of cone bipolar neurons. These types correspond to those identified by the Golgi method and by intracellular recording. Those bipolar cell types for which we reconstructed three or four examples were extremely regular in form, size, and cytology, and also in the quantitative details of their synaptic connections. They appeared quite as specific in these respects as invertebrate "identified" neurons. The synaptic patterns observed for each type of bipolar neuron were complex but may be summarized as follows: the rod bipolar axon ended in sublamina b of the inner plexiform layer and provided major input to the AII amacrine cell. The axons of three types of cone bipolar cells also terminated in sublamina b and provided contacts to dendrites of on-beta and other ganglion cells. All three types, but especially the Cb1, received gap junction contacts from the AII amacrine cell. Axons of four types of cone bipolar cells terminated in sublamina a of the inner plexiform layer and contacted dendrites of off-beta and other ganglion cells. One of these cone bipolar cell types, CBa1, made reciprocal chemical contacts with the lobular appendage of the AII amacrine cell. These results show that the pattern of cone bipolar cell input to beta (X) and probably alpha (Y) ganglion cells is substantially more complex than had been suspected. At least two types of cone bipolar contribute to each type of ganglion cell where only a single type had been anticipated. In addition, many of the cone bipolar cell pathways in the inner plexiform layer are available to the rod system, since at least four types of cone bipolar receive electrical or chemical inputs from the AII amacrine cell. This may help to explain why, in a retina where rods far outnumber the cones, there should be so many types of cone bipolar cells.     

Calretinin in neurochemically well-defined cell populations of rabbit retina

In the rabbit retina, parvalbumin has been localized selectively to AII amacrine cells, while 28 kDa calbindin could be detected in horizontal cells, in one type of depolarizing cone bipolar cell and a population of wide-field amacrine cells. The distribution of the third neuronal calcium binding protein, calretinin, however, has not been studied to date in detail in the rabbit retina. Therefore in this study we aimed to describe the overall distribution of calretinin in the different retinal layers and the possible colocalization pattern with other neurochemical marker molecules. A few cone photoreceptor cells were found to be labeled, whereas the outer plexiform layer was free from immunoreactive elements. In the most proximal row of the inner nuclear layer amacrine cells were labeled, while more distally a few cells emitted beaded axon-like processes, toward the outer retina. There were large (18–28 μm in diameter) cells labeled in the ganglion cell layer, of which many apparently had their axon stained. Some of the calretinin immunoreactive amacrine cells (the AII neurons) also contained parvalbumin. Colocalization of calretinin and 28 kDa calbindin could not be ascertained in the same amacrine cell populations, nor was tyrosine hydroxylase present in calretinin-containing cells. There was partial colocalization of calretinin in the γ-aminobutyric acid-positive amacrine cell population. Parvalbumin containing ganglion cells were also positive for calretinin; however, the calretinin-positive ganglion cells were more numerous. γ-Aminobutyric acid could be colocalized in some calretinin-positive neurons of the ganglion cell layer.