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 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.     

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.     

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.     

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.     

Localization of glycine receptor alpha subunits on bipolar and amacrine cells in primate retina

The major inhibitory neurotransmitter glycine is used by about half of the amacrine cells in the retina. Amacrine cells provide synaptic output to bipolar, ganglion, and other amacrine cells. The present study investigated whether different bipolar and amacrine cell types in the primate retina differ with respect to the expression of glycine receptor (GlyR) subtypes. Antibodies specific for the alpha1, alpha2, and alpha3 subunits of the GlyR were combined with immunohistochemical markers for bipolar and amacrine cells and applied to vertical sections of macaque (Macaca fascicularis) and marmoset (Callithrix jacchus) retinae. For all subunits, punctate immunoreactivity was expressed in the inner plexiform layer. The GlyRalpha2 immunoreactive (IR) puncta occur at the highest density, followed by GlyR(alpha)3 and GlyR(alpha)1 IR puncta. Postembedding electron microscopy showed the postsynaptic location of all subunits. Double immunofluorescence demonstrated that the three alpha subunits are clustered at different postsynaptic sites. Two OFF cone bipolar cell types (flat midget and diffuse bipolar DB3), are predominantly associated with the alpha1 subunit. Two ON bipolar cell types, the DB6 and the rod bipolar cell, are predominantly associated with the alpha2 subunit. The glycinergic AII amacrine cell is presynaptic to the alpha1 subunit in the OFF-sublamina, and postsynaptic to the alpha2 subunit in the ON-sublamina. Another putative glycinergic cell, the vesicular glutamate transporter 3 cell, is predominantly presynaptic to the alpha2 subunit. The dopaminergic amacrine cell expresses the alpha3 subunit at a low density.     

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.     

Connectivity between the OFF bipolar type DB3a and six types of ganglion cell in the marmoset retina

Parallel visual pathways originate at the first synapse in the retina, where cones make connections with cone bipolar cells that in turn contact ganglion cells. There are more ganglion cell types than bipolar types, suggesting that there must be divergence from bipolar to ganglion cells. Here we analyze the contacts between an OFF bipolar type (DB3a) and six ganglion cell types in the retina of the marmoset monkey (Callithrix jacchus). Ganglion cells were transfected via particle‐mediated gene transfer of an expression plasmid for the postsynaptic density 95‐green fluorescent protein (PSD95‐GFP), and DB3a cells were labeled via immunohistochemistry. Ganglion cell types that fully or partially costratified with DB3a cells included OFF parasol, OFF midget, broad thorny, recursive bistratified, small bistratified, and large bistratified cells. On average, the number of DB3a contacts to parasol cells (18 contacts per axon terminal) is higher than that to other ganglion cell types (between four and seven contacts). We estimate that the DB3a output to OFF parasol cells accounts for at least 30% of the total DB3a output. Furthermore, we found that OFF parasol cells receive approximately 20% of their total bipolar input from DB3a cells, suggesting that other diffuse bipolar types also provide input to OFF parasol cells. We conclude that DB3a cells preferentially contact OFF parasol cells but also provide input to other ganglion cell types. J. Comp. Neurol. 524:1839–1858, 2016. © 2015 Wiley Periodicals, Inc.     

GABA-ergic and glycinergic pathways in the inner plexiform layer of the goldfish retina

GABA-ergic and glycinergic circuitry in the inner plexiform layer of the goldfish retina was evaluated by electron microscopic autoradiography of 3H-GABA and 3H-glycine uptake, combined with retrograde horseradish peroxidase (HRP) labeling of ganglion cells. GABA-ergic and glycinergic synapses were found on labeled ganglion cells throughout the inner plexiform layer. This reinforces the idea that physiological evidence of GABA-ergic and glycinergic influence on a variety of ganglion cells in goldfish and carp often reflects direct inputs. Double-labeled synapses are presented as evidence of direct type Ab amacrine cell input to on-center ganglion cells. At least one population of type Aa sustained-off GABA-ergic amacrine cell is proposed, on the basis of profuse GABA-ergic inputs onto bipolar cells in sublamina a. Similar GABA-labeled profiles are shown to synapse onto HRP-labeled probable off-center ganglion cells. Thus GABA-ergic amacrine cells not only provide the predominant feedback to depolarizing (on-center) and hyperpolarizing (off-center) bipolar cells but also provide feed-forward inputs to on- and off-center ganglion cells. Large-caliber GABA-ergic dendrites present in both sublaminae a and b resemble those expected of a previously described bistratified, transient amacrine cell. These processes synapse onto HRP-labeled ganglion cell profiles in both sublaminae. Two morphologies of glycinergic amacrine cell are proposed on the basis of light microscopic autoradiography, 1) the previously described small pyriform cell and 2) a multipolar cell. The differential connectivity of the glycinergic neurons described, however, remains indistinguishable. Whereas abundant glycinergic inputs to ganglion cells occur throughout the inner plexiform layer, contacts between glycinergic profiles and bipolar cells are extremely rare. Therefore, interpreting the meaning of glycinergic input to ganglion cells will require further study of amacrine cell circuitry.     

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.     

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.     

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.     

Identification of AⅡ amacrine,displaced amacrine,and bistratified ganglion cell types in human retina with antibodies against calretinin

Antibodies against calretinin are markers for one type of rod pathway interneuron (AⅡ amacrine cell) in the retina of some but not all mammalian species. The AⅡ cells play a crucial role in night‐time (scotopic) vision and have been proposed as a target for optogenetic restoration of vision in retinal disease. In the present study we aimed to characterize the AⅡ cells in human retina. Postmortem human donor eyes were obtained with ethical approval and processed for calretinin immunofluorescence. Calretinin‐positive somas in the inner nuclear and the ganglion cell layer were filled with the lipophilic dye DiI. The large majority (over 80%) of calretinin‐immunoreactive cells is located in the inner nuclear layer, is immunopositive for glycine transporter 1, and shows the typical morphology of AⅡ amacrine cells. In addition, a small proportion of calretinin‐positive cells in the inner nuclear layer and in the ganglion cell layer is glutamic acid decarboxylase‐positive and shows the morphology of widefield amacrine cells (stellate, semilunar, and thorny amacrine cells). About half of the calretinin cells in the ganglion cell layer are bistratified ganglion cells resembling the small bistratified (presumed blue‐ON/yellow‐OFF) and the G17 ganglion cell previously described in primates. We conclude that in human retina, antibodies against calretinin can be used to identify AⅡ amacrine cells in the inner nuclear layer as well as widefield amacrine and small bistratified ganglion cells in the ganglion cell layer. J. Comp. Neurol. 524:39–53, 2016. © 2015 Wiley Periodicals, Inc.     

Type 3a and type 3b OFF cone bipolar cells provide for the alternative rod pathway in the mouse retina

The mammalian retina provides several pathways to relay the information from the photoreceptors to the ganglion cells. Cones feed into ON and OFF cone bipolar cells that excite ON and OFF ganglion cells, respectively. In the "classical" rod pathway, rods feed into rod bipolar cells that provide input to both the ON and the OFF pathway via AII amacrine cells. Recent evidence suggests an alternative rod pathway in which rods directly contact some types of OFF cone bipolar cells. The mouse has become an important model system for retinal research. We performed an immunohistochemical analysis on the level of light and electron microscopy to identify the bipolar cells and ganglion cells that are involved in the alternative rod pathway of the mouse retina. 1) We identify a new bipolar cell type, showing that type 3 OFF cone bipolar cells comprise two distinct cell types, that we termed 3a and 3b. Type 3a cells express the ion channel HCN4. Type 3b bipolar cells represent a hitherto unknown cell type that can be identified with antibodies against the regulatory subunit RIIbeta of protein kinase A. 2) We show that both 3a and 3b cells form flat contacts at cone pedicles and rod spherules. 3) Finally, we identify an OFF ganglion cell type whose dendrites costratify with type 3a and 3b bipolar cell axon terminals. These newly identified cell types represent the basis of a neuronal circuit in the mammalian retina that could provide for an alternative fast rod pathway.     

Glycine receptor-mediated synaptic transmission regulates the maturation of ganglion cell synaptic connectivity

It is well documented that neuronal activity is required for the developmental segregation of retinal ganglion cell (RGC) synaptic connectivity with ON and OFF bipolar cells in mammalian retina. Our recent study showed that light deprivation preferentially blocked the developmental RGC dendritic redistribution from the center to sublamina a of the inner plexiform layer (IPL). To determine whether OFF signals in visual stimulation are required for OFF RGC dendritic development, the light-evoked responses and dendritic stratification patterns of RGCs in Spastic mutant mice, in which the OFF signal transmission in the rod pathway is largely blocked due to a reduction of glycine receptor (GlyR) expression, were quantitatively studied at different ages and rearing conditions. The dendritic distribution in the IPL of these mice was indistinguishable from wildtype controls at the age of postnatal day (P)12. However, the adult Spastic mutants had altered RGC light-evoked synaptic inputs from ON and OFF pathways, which could not be mimicked by pharmacologically blocking of glycinergic synaptic transmission on age-matched wildtype animals. Spastic mutation also blocked the developmental redistribution of RGC dendrites from the center to sublamina a of the IPL, which mimicked the effects induced by light deprivation on wildtype animals. Moreover, light deprivation of the Spastic mutants had no additional impact on the RGC dendritic distribution and light response patterns. We interpret these results as that visual stimulation regulates the maturation of RGC synaptic activity and connectivity primarily through GlyR-mediated synaptic transmission.     

Localization of heterotypic gap junctions composed of connexin45 and connexin36 in the rod pathway of the mouse retina

The primary rod pathway in mammals contains gap junctions between AII amacrine cells and ON cone bipolar cells which relay the rod signal into the cone pathway under scotopic conditions. Two gap junctional proteins, connexin36 (Cx36) and connexin45 (Cx45), appear to play a pivotal role in this pathway because lack of either protein leads to an impairment of visual transmission under scotopic conditions. To investigate whether these connexins form heterotypic gap junctions between ON cone bipolar and AII amacrine cells, we used newly developed Cx45 antibodies and studied the cellular and subcellular distribution of this protein in the mouse retina. Specificity of the Cx45 antibodies was determined, among others, by Western blot and immunostaining of mouse heart, where Cx45 is abundantly expressed. In mouse retina, Cx45 immunosignals were detected in both plexiform layers and the ganglion cell layer. Double staining for Cx45 and Cx36 revealed a partial overlap in the punctate patterns in the ON sublamina of the inner plexiform layer of the retina. We quantified the distributions of these two connexins in the ON sublamina, and detected 30% of the Cx45 signals to be co-localized with or in close apposition to Cx36 signals. Combining immunostaining and intracellular dye injection revealed an overlap or tight association of Cx36 and Cx45 signals on the terminals of injected AII amacrine and two types of ON cone bipolar cells. Our results provide direct evidence for heterotypic gap junctions composed of Cx36 and Cx45 between AII amacrine and certain types of ON cone bipolar cells.     

Off-alpha and OFF-beta ganglion cells in cat retina: II. Neural circuitry as revealed by electron microscopy of HRP stains

An OFF-center alpha and an OFF-center beta ganglion cell in cat retina, which had been recorded from and intracellularly stained with horseradish peroxidase (HRP) were examined by serial section electron microscopy. We counted synapses and identified presynaptic neurons to the HRP-stained cells in 20 μm radial slices through the centers of their dendritic trees. Presynaptic amacrine and bipolar cells were identified on cytological criteria known from previous studies. The OFF-beta cell with a 62 μm dendritic arbor, restricted to S1 and S2 (sublamina a) of the inner plexiform layer (IPL), received 38% bipolar and 62% amacrine cell synapses. The bipolar input was from both cb1 and cb2 cone bipolar types. Input from three distinct amacrine cell types occurred upon the dendrites, namely from: (1) AII amacrine lobular appendages, (2) large pale amacrine profiles (possibly A2 or A3 cells), and (3) small, dark amacrine types (possibly A8 cells). Large pale amacrine profiles (possibly A13) were found on the cell body and apical dendrite in sublamina b of the IPL. In addition, several amacrine profiles synapsed directly on the sides and base of the cell body in the ganglion cell layer. We estimate that the complete dendritic tree of this beta cell received about 1,000 synapses contributed by 12–14 bipolar cells, 7–10 AII amacrines and 28–41 other amacrine cells. The OFF-alpha cell had a dendritic tree size of 680 × 920 μm. A 250 μm length of two major dendrites stratifying narrowly in S2 of the IPL was reconstructed. Amacrine cells provided most of the synaptic input (80%). This input came from: (1) AII amacrine lobular appendages, (2) amacrines exhibiting large, pale synaptic profiles (possibly A2 or A3 cells), (3) pale amacrines with large mitochondria and a few neurotubules (unknown type), and (4) densely neurotubule-filled amacrine profiles (possibly A19 cells). A large pale amacrine cell type (possibly A13) provided synaptic input to the cell body as a serial synaptic intermediary with rod bipolar cells. Cone bipolar synapses were from only one type of cone bipolar, the cb2 type and formed 20% of the total synaptic input. We estimate that a minimum of 142 bipolar cells, 256 AII amacrine cells and 1,011 other amacrine cells, altogether providing 6,000–10,000 synapses, converged on the dendritic tree of this OFF-alpha cell. © 1993 Wiley-Liss, Inc.     

Glutamatergic input is coded by spike frequency at the soma and proximal dendrite of AII amacrine cells in the mouse retina

In the mammalian retina, AII amacrine cells play a crucial role in scotopic vision. They transfer rod signals from rod bipolar cells to the cone circuit, and divide these signals into the ON and OFF pathways at the discrete synaptic layers. AII amacrine cells have been reported to generate tetrodotoxin (TTX)-sensitive repetitive spikes of small amplitude. To investigate the properties of the spikes, we performed whole-cell patch-clamping of AII amacrine cells in mouse retinal slices. The spike frequency increased in proportion to the concentration of glutamate puffer-applied to the arboreal dendrite and to the intensity of the depolarizing current injection. The spike activity was suppressed by L-2-amino-4-phosphonobutyric acid, a glutamate analogue that hyperpolarizes rod bipolar cells, puffer-applied to the outer plexiform layer. Therefore, it is most likely that the spike frequency generated by AII amacrine cells is dependent on the excitatory glutamatergic input from rod bipolar cells. Gap junction blockers reduced the range of intensity of input with which spike frequency varies. Application of TTX to the soma and the proximal dendrite of AII amacrine cells blocked the voltage-gated Na(+) current significantly more than application to the arboreal dendrite, indicating that the Na(+) channels are mainly localized in these regions. Our results suggest that the intensity of the glutamatergic input from rod bipolar cells is coded by the spike frequency at the soma and the proximal dendrite of AII amacrine cells, raising the possibility that the spikes could contribute to the OFF pathway to enhance release of neurotransmitter.     

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

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

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.