Diversity of glycine receptors in the mouse retina: localization of the alpha2 subunit

Gamma-aminobutyric acid (GABA) and glycine are the major inhibitory neurotransmitters in the retina, glycine being produced in approximately half of all amacrine cells. Whereas retinal cell types expressing the glycine receptor (GlyR) alpha1 and alpha3 subunits have been mapped, the role of the alpha2 subunit in retinal circuitry remains unclear. By using immunocytochemistry, we localized the alpha2 subunit in the inner plexiform layer (IPL) in brightly fluorescent puncta, which represent postsynaptically clustered GlyRs. This was shown by doubly labeling sections for GlyR alpha2 and bassoon (a presynaptic marker) or gephyrin (a postsynaptic marker). Synapses containing GlyR alpha2 were rarely found on ganglion cell dendrites but were observed on bipolar cell axon terminals and on amacrine cell processes. Recently, an amacrine cell type has been described that is immunopositive for glycine and for the vesicular glutamate transporter vGluT3. The processes of this cell type were presynaptic to GlyR alpha2 puncta, suggesting that vGluT3 amacrine cells release glycine. Double labeling of sections for GlyR alpha1 and GlyR alpha2 subunits showed that they are clustered at different synapses. In sections doubly labeled for GlyR alpha2 and GlyR alpha3, approximately one-third of the puncta were colocalized. The most abundant GlyR subtype in retina contains alpha3 subunits, followed by those containing GlyR alpha2 and GlyR alpha1 subunits.     

Diversity of glycine receptors in the mouse retina: localization of the alpha4 subunit

Glycine and gamma-aminobutyric acid (GABA) are the major inhibitory neurotransmitters in the retina. Approximately half of the amacrine cells release glycine at their synapses with bipolar, other amacrine, and ganglion cells. Whereas the retinal distributions of glycine receptor (GlyR) subunits alpha1, alpha2, and alpha3 have been mapped, the role of the alpha4 subunit in retinal circuitry remains unclear. A rabbit polyclonal antiserum was raised against a peptide that comprises the C-terminal 14 amino acids of the mouse GlyR alpha4 subunit. Using immunocytochemistry, we localized the alpha4 subunit in the inner plexiform layer (IPL) in brightly fluorescent puncta, which represent postsynaptically clustered GlyRs. This was shown by double-labeling sections for GlyR alpha4 and synaptic markers (bassoon, gephyrin). Double-labeling sections for GlyR alpha4 and the other GlyR alpha subunits shows that they are mostly clustered at different synapses; however, approximately 30% of the alpha4-containing synapses also express the alpha2 subunit. We also studied the pre- and postsynaptic partners at GlyR alpha4-containing synapses and found that displaced (ON-) cholinergic amacrine cells prominently expressed the alpha4 subunit. The density of GlyR alpha4-expressing synapses in wildtype, Glra1(ot/ot), and Glra3(-/-) mouse retinas did not differ significantly. Thus, there is no apparent compensation of the loss of alpha1 or alpha3 subunits by an upregulation of alpha4 subunit gene expression; however, the alpha2 subunit is moderately upregulated.     

Diversity of glycine receptors in the mouse retina: localization of the alpha3 subunit

Glycine receptors (GlyRs) and their role in retinal circuitry were analyzed immunocytochemically in wild-type and GlyR alpha3 subunit-deficient (Glra3(-/-)) mouse retinae. GlyRs are localized in the inner plexiform layer in brightly fluorescent puncta, which are likely to represent postsynaptically clustered GlyRs. Approximately one third of the clusters were found to contain the alpha1 subunit, and half possessed the alpha3 subunit. However, these two GlyR isoforms were localized at different glycinergic synapses. In the Glra3(-/-) mouse, alpha3 subunit clusters were completely eliminated, although the total number of GlyR clusters was only slightly reduced. This finding indicates that other GlyR subunits (such as alpha2 or alpha4) may have compensated for the loss of the alpha3 subunit. Characteristic expression patterns of the alpha1 and alpha3 subunits within the synaptic circuits of the retina were revealed by double labeling sections for GlyRs and markers that define specific retinal neurons. The alpha1 subunit mediates signal transfer in the rod pathway between AII amacrine cells and OFF-cone bipolar cells. In contrast, the alpha3 subunit appears to be predominantly involved with the cone pathways. Thus, expression of different GlyR alpha subunit genes correlates with anatomically defined connectivities.     

Characterization of the glycinergic input to bipolar cells of the mouse retina

Glycine and gamma-aminobutyric acid (GABA) are the major inhibitory transmitters of the mammalian retina, and bipolar cells receive GABAergic and glycinergic inhibition from multiple amacrine cell types. Here we evaluated the functional properties and subunit composition of glycine receptors (GlyRs) in bipolar cells. Patch-clamp recordings were performed from retinal slices of wild-type, GlyRalpha1-deficient (Glra1(spd-ot)) and GlyRalpha3-deficient (Glra3(-/-)) mice. Whole-cell currents following glycine application and spontaneous inhibitory postsynaptic currents (IPSCs) were analysed. During the recordings the cells were filled with Alexa 488 and, thus, unequivocally identified. Glycine-induced currents of bipolar cells were picrotoxinin-insensitive and thus represent heteromeric channels composed of alpha and beta subunits. Glycine-induced currents and IPSCs were absent from all bipolar cells of Glra1(spd-ot) mice, indicating that GlyRalpha1 is an essential subunit of bipolar cell GlyRs. By comparing IPSCs of bipolar cells in wild-type and Glra3(-/-) mice, no statistically significant differences were found. OFF-cone bipolar (CB) cells receive a strong glycinergic input from AII amacrine cells, that is preferentially based on the fast alpha1beta-containing channels (mean decay time constant tau = 5.9 +/- 1.4 ms). We did not observe glycinergic IPSCs in ON-CB cells and could elicit only small, if any, glycinergic currents. Rod bipolar cells receive a prominent glycinergic input that is mainly mediated by alpha1beta-containing channels (tau = 5.5 +/- 1.6 ms). Slow IPSCs, the characteristic of GlyRs containing the alpha2 subunit, were not observed in bipolar cells. Thus, different bipolar cell types receive kinetically fast glycinergic inputs, preferentially mediated by GlyRs composed of alpha1 and beta subunits.     

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.     

Distribution of glycine receptor subunits on primate retinal ganglion cells: a quantitative analysis

This study investigates the distribution of inhibitory neurotransmitter receptors on sensory neurons. Ganglion cells in the retina of a New World monkey, the common marmoset Callithrix jacchus, were injected with Lucifer yellow and Neurobiotin and subsequently processed with antibodies against one (alpha1), or against all subunits, of the glycine receptor, or against the anchoring protein gephyrin. Immunoreactive (IR) puncta representing glycine receptor or gephyrin clusters were found on the proximal and the distal dendrites of all ganglion cell types investigated. For both parasol and midget cells, the density of receptor clusters was greater on distal than proximal dendrites for all antibodies tested. In parasol cells the average density for the alpha1 subunit of the glycine receptor was 0.087 IR puncta/microm of dendrite, and for all subunits it was 0.119 IR puncta/microm of dendrite. Thus, the majority of glycine receptors on parasol cells contain the alpha1 subunit. For parasol cells, we estimated an average of 1.5 glycinergic synapses/100 microm2 dendritic membrane on proximal dendrites and about 9.4 glycinergic synapses/100 microm2 on distal dendrites. The segregation of receptors to the distal dendrites appears to be a common feature of inhibitory neurotransmitter input to parasol and midget cells, and might be associated with the receptive field surround mechanism.     

Glutamate receptors at bipolar synapses in the inner plexiform layer of primate retina: light microscopic analysis

At least 10 different types of bipolar cells have been distinguished in the primate retina. The axon terminals of these cells stratify in distinct strata in the inner plexiform layer and are involved in parallel pathways to distinct types of ganglion cells. Ionotropic glutamate receptor (GluR) subunits also show a stratified distribution in the inner plexiform layer. Here, we investigated whether different types of bipolar cells are associated with different types of ionotropic glutamate receptors in the inner retina of a New World primate, the common marmoset Callithrix jacchus. Vertical cryostat sections through central retina were double labeled with immunohistochemical markers for bipolar cell types and with antibodies to alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunits GluR1 to 4, kainate receptor subunits GluR6/7, and the NR1C2' subunit of the N-methyl-D-aspartate (NMDA) receptor. The axon terminals of bipolar cell types were reconstructed from confocal sections, and the colocalized immunoreactive puncta were quantified. For all bipolar cell types, immunoreactive puncta for the AMPA receptor subunits GluR2, 2/3, and 4 were colocalized at highest densities, whereas GluR1-immunoreactive puncta were expressed at very low densities. The kainate receptor subunits GluR6/7 were predominantly associated with diffuse bipolar (DB6) and rod bipolar cells. The NMDA receptor subunit NR1C2' was specifically colocalized with flat midget and DB3 axons. These findings suggest that rod and cone bipolar cell types contribute to multiple but distinct glutamate receptor pathways in primate retina.     

GABAA and GABAC receptors on mammalian rod bipolar cells

Rod bipolar (RB) cells of mammalian retinae receive synapses from different γ-aminobutyric acid (GABAergic) amacrine cells in the inner plexiform layer (IPL). We addressed the question whether RB cells of the rabbit and of the rat retina express different types of GABA receptors at these synapses. RB cells were immunolabeled in vertical sections of rat retinae with an antibody against protein kinase C (PKC). The sections were double-labled for the α1, α2, α3, or γ2 subunits of the GABA_A receptor. Punctate immunofluorescence, which represents synaptic localization, was found for all four subunits. Many of the α1-, α3-, or γ2-immunoreactive puncta coincided with the axon terminals of the PKC-immunolabeled RB cells. Sections and wholemounts of rabbit retinae were also double labeled for PKC and the ρ subunits of the GABA_C receptor. Rabbit RB cells were decorated by many ρ-immunoreactive puncta, which were shown by electron microscopy to represent synaptic localization. Previous work from our laboratory has shown that the α1, α2, α3, and ρ subunits are not found within the same synapse but are expressed at different synaptic sites. Taken together, these results suggest that RB cells of mammalian retinae express at least three different types of GABA receptors at synaptic sites in the IPL: GABA_C receptors, GABA_Areceptors containing the α1 subunit, and GABA_A receptors containing the α3 subunit. J. Comp. Neurol. 396:351–365, 1998. © 1998 Wiley-Liss, Inc.     

Glycine-receptor immunoreactivity in retinal bipolar cells is postsynaptic to glycinergic and GABAergic amacrine cell synapses.

Glycinergic innervation of the synaptic terminals of mixed rod-cone bipolar cells in the goldfish retina was investigated by electron microscopical immunocytochemistry with presynaptic and postsynaptic markers for glycinergic neurons: a monoclonal antibody (mAb 7A) against the 93 kDa subunit of the strychnine-sensitive glycine receptor and polyclonal antisera against a glycine/BSA conjugate. Conventional "glycinergic" synaptic contacts, made by amacrine cell processes, accounted for 7-10% of the input to the bipolar cell terminals, whether determined by glycine receptor immunoreactivity (GlyR-IR) or glycine-IR. In addition to the conventional synapses, the large bipolar cell terminals in the proximal inner plexiform layer (type Mb) gave rise to spinules (spine-like protrusions) that invaginated into presynaptic amacrine cell processes. Although 85% of the spinules were GlyR-IR, no spinules were postsynaptic to glycine-IR processes; yet 86% of the spinules were postsynaptic to GAD-IR processes, suggesting that the GlyR-IR spinules were postsynaptic to GABAergic terminals. Furthermore, a single amacrine cell process could make two synapses with an Mb terminal: a GlyR-IR contact onto a spinule and a conventional synapse that was not GlyR-IR. We suggest that glycinergic innervation of bipolar cell terminals involves conventional glycinergic synapses as well as an unconventional situation in which GABA and glycine may interact in as yet undetermined manner, perhaps by potentiation.     

Immunohistochemical identification and synaptic inputs to the diffuse bipolar cell type DB1 in macaque retina

Detailed analysis of the synaptic inputs to the primate DB1 bipolar cell has been precluded by the absence of a suitable immunohistochemical marker. Here we demonstrate that antibodies for the EF-hand calcium-binding protein, secretagogin, strongly label the DB1 bipolar cell as well as a mixed population of GABAergic amacrine cells in the macaque retina. Using secretagogin as a marker, we show that the DB1 bipolar makes synaptic contact with both L/M as well as S-cone photoreceptors and only minimal contact with rod photoreceptors. Electron microscopy showed that the DB1 bipolar makes flat contacts at both triad-associated and nontriad-associated positions on the cone pedicle. Double labeling with various glutamate receptor subunit antibodies failed to conclusively determine the subunit composition of the glutamate receptors on DB1 bipolar cells. In the IPL, DB1 bipolar cell axon terminals expressed the glycine receptor, GlyRα1, at sites of contact with AII amacrine cells, suggesting that these cells receive input from the rod pathway.     

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

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

Glycinergic input of small-field amacrine cells in the retinas of wildtype and glycine receptor deficient mice

Amacrine cells are known to express strychnine-sensitive glycine receptors (GlyRs), however, it is not known which of the four GlyRalpha subunits (alpha1-4) are expressed in this diverse group of cells. Herein, we studied the presence of glycine activated currents and spontaneous inhibitory postsynaptic currents (sIPSCs) of amacrine cells in the mouse retina. By recording glycinergic currents in retinal slices of wildtype mice and of mice deficient in GlyRalpha subunits (Glra1spd-ot, Glra2-/-, Glra3-/-), we could classify AII and narrow-field amacrine cells (NF, Types 5, 6, 7) on the basis of their alpha-subunit composition. Glycinergic sIPSCs of AII cells displayed medium fast kinetics (mean decay time constant tau=11+/-2 ms), which were completely absent in the Glra3-/- mouse, indicating that synaptic GlyRs of AII cells mainly contain the alpha3 subunit. Glycinergic sIPSCs of NF cells had slow kinetics (tau=27+/-6.8 ms) that were significantly prolonged in Glra2-/- mice (tau=69+/-16 ms). These data show that morphologically distinct amacrine cells express different sets of GlyRs.     

Indoleamine-accumulating amacrine cells are presynaptic to rod bipolar cells through GABA(C) receptors.

gamma-Aminobutyric acid (GABA), is a main source of inhibitory modulation of the rod pathway in the mammalian retina. The authors previously showed that rod bipolar cells express at least three types of ionotropic GABA receptors. Here, the authors sought to determine which neurons are the presynaptic partners at these synapses in the rabbit retina. Indoleamine-accumulating amacrine cells (IACs) were immunolabeled with an antiserum against serotonin (5HT) in vertical sections and wholemounts of rabbit retinae that had been preloaded with 5HT. The tissue was double labeled for the rho subunits of the GABA(C) receptor or the alpha3 subunit of the GABA(A) receptor. Punctate immunofluorescence was observed for both receptor subunits and was found to coincide with the dendrites and varicosities of IACs. The localization of rho subunits was examined at the ultrastructural level by using postembedding techniques on slam-frozen, cryosubstituted tissue. Double labeling at the electron microscopic level revealed that 5HT-immunoreactive processes were presynaptic to rod bipolar cells through GABA(C) receptors. Intracellular injection of the two morphologic subclasses of IAC amacrine cells, S1 and S2, with Lucifer yellow followed by immunolabeling for the alpha3 or rho subunits revealed that varicosities on the dendrites of both cell types were in register with alpha3- and rho-immunoreactive puncta. Taken together, these results suggest that IACs are presynaptic to rod bipolar cells through GABA(C) receptors and possibly through GABA(A) receptors.     

Glycinergic interplexiform cells make synaptic contact with amacrine cell bodies in goldfish retina.

Recent works utilizing glycine-immunoreactivity (IR) and combined Golgi impregnation and 3H-glycine uptake autoradiography indicate that glycinergic interplexiform cells (IPC) may synapse upon cell bodies in the inner nuclear and ganglion cell layers in fish retina. This possibility was investigated with immunocytochemical techniques using presynaptic and postsynaptic markers for glycinergic neurons: a monoclonal antibody (mAb 7A) against the 93 kDa subunit of the strychnine-sensitive glycine receptor and a polyclonal antiserum against a glycine/BSA conjugate. Synaptic contacts onto the lateral and proximal surfaces of amacrine cell bodies and onto the distal surface of cells in the ganglion cell layer were identified with both probes. The contacts were rare with one contacted amacrine cell/section of 500 linear micron. Serial 1-micron sections were processed alternately for glycine and GABA antisera using postembedding techniques at the light microscopic level. Glycine-IR processes + boutons were apposed to GABA-IR cell bodies in 16 of 17 examples, indicating that the dendro-somatic contacts were onto GABA-immunoreactive amacrine cell bodies. In context of other published morphological data, we suggest that the dendro-somatic synapses were derived from glycinergic IPCs. Glycinergic IPCs receive input from GABAergic horizontal cells and, via a shunt conductance produced by the dendro-somatic contacts, may be involved in controlling the sensitivity, temporal, or spatial properties of amacrine cell responses to large field illumination.     

Synaptic distribution of ionotropic glutamate receptors in the inner plexiform layer of the primate retina

The distribution and synaptic clustering of glutamate receptors (GluRs) were studied in the inner plexiform layer (IPL) of the macaque monkey retina by using subunit specific antisera. A punctate immunofluorescence pattern was observed in the IPL for all subunits tested, and electron microscopy confirmed that the immunoreactive puncta represent clustering of receptors at sites postsynaptic to the bipolar cell ribbon synapses (dyads). Usually only one of the two postsynaptic processes at the dyads expressed a given subunit. Immunoreactive GluR2, GluR2/3, and GluR4 puncta were found at high density throughout the IPL and are probably expressed at every dyad. The GluR1 subunit was expressed at lower density. The N-methyl-D-aspartate (NMDA) receptor subunits NR2A and NR1C2' were restricted to synapses localized in two broad bands in the center of the IPL. They were often colocalized with GluR2/3 and GluR4 subunits. The orphan receptor subunits delta 1/2 predominated in three horizontal bands. The kainate receptor subunits GluR6/7 were clustered in large postsynaptic densities adjacent to bipolar cell axon terminals but lacking a synaptic ribbon on the presynaptic side. This might represent a conventional synapse made by a bipolar axon terminal. The results suggest that GluR2/3 and GluR4, together with NMDA receptors, are preferentially expressed on ganglion cell dendrites, whereas kainate receptors and the delta 1/2 subunits are mostly localized on amacrine cell processes.     

Immunocytochemical localization of glycine receptors in the mammalian retina

The distribution of glycinergic synapses in the mammalian retina was studied with monoclonal antibodies against glycine receptors and a glycine receptor-related protein (gephyrin). Monoclonal antibody 2b is specific for the α1 subunit of the glycine receptor; monoclonal antibody 4a is specific for all known α subunits and the β subunit, and monoclonal antibody 7a is specific for gephyrin. The three antibodies were applied to the retina of cat, macaque monkey, rat, and rabbit. The general staining pattern is comparable in all these species and it is similar but distinct with all of the three antibodies. Labeling is characterized by a punctate appearance indicating that it occurs at synapses. In the inner plexiform layer, labeling is concentrated in two bands. One band is located close to the inner nuclear layer; the other band is located in the middle of the inner plexiform layer. In the outer plexiform layer, sparse punctate labeling is seen. The distribution of gephyrin was also studied at the ultrastructural level in cat and monkey retina. Gephyrin is present on the postsynaptic membrane of amacrine cells and ganglion cells. The presynaptic profile to gephyrin immunoreactivity is always of an amacrine cell. The AII amacrine cell, the crucial glycinergic interneuron of the rod pathway, is presynaptic to gephyrin immunoreactivity in the OFF-sublamina and is itself gephyrin-positive at an input synapse from another (possibly GABAergic) amacrine cell in the ON-sublamina. © 1993 Wiley-Liss, Inc.     

Association of the AMPA receptor-related postsynaptic density proteins GRIP and ABP with subsets of glutamate-sensitive neurons in the rat retina

We used specific antibodies against two postsynaptic density proteins, GRIP (glutamate receptor interacting protein) and ABP (AMPA receptor-binding protein), to study their distribution in the rat retina. In the central nervous system, it has been shown that both proteins bind strongly to the AMPA glutamate receptor (GluR) 2/3 subunits, but not other GluRs, through a set of three PDZ domains. Western blots detected a single GRIP protein that was virtually identical in retina and brain, whereas retinal ABP corresponded to only one of three ABP peptides found in brain. The retinal distributions of GluR2/3, GRIP, and ABP immunoreactivity (IR) were similar but not identical. GluR2/3 immunoreactivity (IR) was abundant in both plexiform layers and in large perikarya. ABP IR was concentrated in large perikarya but was sparse in the plexiform layers, whereas GRIP IR was relatively more abundant in the plexiform layers than in perikarya. Immunolabel for these three antibodies consisted of puncta < or = 0.2 microm in diameter. The cellular localization of GRIP and ABP IR was examined by double labeling subclasses of retinal neuron with characteristic marker proteins, e.g., calbindin. GRIP, ABP, and GluR2/3 IR were detected in horizontal cells, dopaminergic and glycinergic AII amacrine cells and large ganglion cells. Immunolabel was absent in rod bipolar and weak or absent in cholinergic amacrine cells. By using the tyramide method of signal amplification, a colocalization of GluR2/3 was found with either GRIP or ABP in horizontal cell terminals, and perikarya of amacrine and ganglion cells. Our results show that ABP and GRIP colocalize with GluR2/3 in particular subsets of retinal neuron, as was previously established for certain neurons in the brain.     

Mirror-symmetrical populations of wide-field amacrine cells of the macaque monkey retina

Retinas of macaque monkeys were immunostained for glycogen phosphorylase (glypho). Glypho was localized to regular and displaced amacrine cells. Their processes occupied two narrow strata within the inner plexiform layer (IPL). The labeling pattern is reminiscent of cholinergic amacrine cells; however, double immunostaining of the retinas for choline acetyltransferase and glypho revealed two different cell populations. Intracellular injection of DiI showed that glypho-immunoreactive amacrine cells are wide-field amacrine cells with straight, radially oriented, and sparsely branched dendrites. The density of the cells increased from approximately 70/mm(2) in the peripheral retina to approximately 700/mm(2) in the central retina. The regular glypho-immunoreactive amacrine cells branch in sublamina 2 of the IPL, where they receive input from OFF-cone bipolar cells. The displaced cells branch in sublamina 3/4 and receive input from ON-cone bipolar cells. This suggests that the regular cells are OFF-cells and the displaced cells are ON-cells. The cells express gamma-aminobutyric acid (GABA)-like immunoreactivity and receive glycinergic input through synapses expressing preferentially the glycine receptor alpha2 subunit. The close proximity of the dendritic strata of glypho-immunoreactive amacrine cells, cholinergic amacrine cells, and direction-selective ganglion cells suggests a possible role of the cells in the generation of direction-selective light responses of the monkey retina.     

Synaptic connections of DB3 diffuse bipolar cell axons in macaque retina

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.     

Glycinergic amacrine cells of the rat retina

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. J. Comp. Neurol. 401:34–46, 1998. © 1998 Wiley-Liss, Inc.