Christian Puller

Cone Contacts, Mosaics, and Territories of Bipolar Cells in the Mouse Retina

We report a quantitative analysis of the different bipolar cell types of the mouse retina. They were identified in wild-type mice by specific antibodies or in transgenic mouse lines by specific expression of green fluorescent protein or Clomeleon. The bipolar cell densities, their cone contacts, their dendritic coverage, and their axonal tiling were measured in retinal whole mounts. The results show that each and all cones are contacted by at least one member of any given type of bipolar cell (not considering genuine blue cones). Consequently, each cone feeds its light signals into a minimum of 10 different bipolar cells. Parallel processing of an image projected onto the retina, therefore, starts at the first synapse of the retina, the cone pedicle. The quantitative analysis suggests that our proposed catalog of 11 cone bipolar cells and one rod bipolar cell is complete, and all major bipolar cell types of the mouse retina appear to have been discovered.

Chromatic Bipolar Cell Pathways in the Mouse Retina

Like most mammals, mice feature dichromatic color vision based on short (S) and middle (M) wavelength-sensitive cone types. It is thought that mammals share a retinal circuit that in dichromats compares S- and M-cone output to generate blue/green opponent signals, with bipolar cells (BCs) providing separate chromatic channels. Although S-cone-selective ON-BCs (type 9 in mouse) have been anatomically identified, little is known about their counterparts, the M-cone-selective OFF-BCs. Here, we characterized cone connectivity and light responses of selected mouse BC types using immunohistochemistry and electrophysiology. Our anatomical data indicate that four (types 2, 3a/b, and 4) of the five mouse OFF-BCs indiscriminately contact both cone types, whereas type 1 BCs avoid S-cones. Light responses showed that the chromatic tuning of the BCs strongly depended on their position along the dorsoventral axis because of the coexpression gradient of M- and S-opsin found in mice. In dorsal retina, where coexpression is low, most type 2 cells were green biased, with a fraction of cells (≈14%) displaying strongly blue-biased responses, likely reflecting S-cone input. Type 1 cells were also green biased but did not comprise blue-biased “outliers,” consistent with type 1 BCs avoiding S-cones. We therefore suggest that type 1 represents the green OFF pathway in mouse. In addition, we confirmed that type 9 BCs display blue-ON responses. In ventral retina, all BC types studied here displayed similar blue-biased responses, suggesting that color vision is hampered in ventral retina. In conclusion, our data support an antagonistically organized blue/green circuit as the common basis for mammalian dichromatic color vision.

Distribution of the glycine receptor β-subunit in the mouse CNS as revealed by a novel monoclonal antibody.

Inhibitory glycine receptors (GlyRs) are composed of homologous α‐ (α1–4) and β‐subunits. The β‐subunits (GlyRβ) interact via their large cytosolic loops with the postsynaptic scaffolding protein gephyrin and are therefore considered essential for synaptic localization. In situ hybridization studies indicate a widespread distribution of GlyRβ transcripts throughout the mammalian central nervous system (CNS), whereas GlyRα mRNAs and proteins display more restricted expression patterns. Here we report the generation of a monoclonal antibody that specifically recognizes rodent GlyRβ (mAb‐GlyRβ) and does not exhibit crossreactivity with any of the GlyRα1–4 subunits. Immunostaining with this antibody revealed high densities of punctate GlyRβ immunoreactivity at inhibitory synapses in mouse spinal cord, brainstem, midbrain, and olfactory bulb but not in the neocortex, cerebellum, or hippocampus. This contrasts the abundance of GlyRβ transcripts in all major regions of the rodent brain and suggests that GlyRβ protein levels are regulated posttranscriptionally. When mAb‐GlyRβ was used in double‐labeling experiments with GlyRα1‐, α2‐, α3‐, or α4‐specific antibodies to examine the colocalization of GlyRβ with these GlyR subunits in the mouse retina, >90% of the GlyRα1–3 clusters detected were found to be GlyRβ‐immunoreactive. A subset (about 50%) of the GlyRα4 puncta in the inner plexiform layer, however, was found to lack GlyRβ and gephyrin immunostaining. These GlyRα4‐only clusters were apposed to bassoon immunoreactivity and hence synaptically localized. Their existence points to a gephyrin‐independent synaptic localization mechanism for a minor subset of GlyRs. J. Comp. Neurol. 520:3962–3981, 2012.

ZO-1 and the Spatial Organization of Gap Junctions and Glutamate Receptors in the Outer Plexiform Layer of the Mammalian Retina

Information processing in the retina starts at the first synaptic layer, where photoreceptors and second-order neurons exhibit a complex architecture of glutamatergic and electrical synapses. To investigate the composition of this highly organized synaptic network, we determined the spatial relationship of zonula occludens-1 (ZO-1) with different connexins (Cx) and glutamate receptor (GluR) subunits in the outer plexiform layer (OPL) of rabbit, mouse, and monkey retinas. ZO-1 is well known as an intracellular component of tight and adherens junctions, but also interacts with various connexins at gap junctions. We found ZO-1 closely associated with Cx50 on dendrites of A-type horizontal cells in rabbit, and with Cx57 at dendro-dendritic gap junctions of mouse horizontal cells. The spatial arrangement of ZO-1 at the giant gap-junctional plaques in rabbit was particularly striking. ZO-1 formed a clear margin around the large Cx50 plaques instead of being colocalized with the connexin staining. Our finding suggests the involvement of ZO-1 in the composition of tight or adherens junctions around gap-junctional plaques instead of interacting with connexins directly. Furthermore, gap junctions were found to be clustered in close proximity to GluRs at the level of desmosome-like junctions, where horizontal cell dendrites converge before invaginating the cone pedicle. Based on this distinct spatial organization of gap junctions and GluRs, it is tempting to speculate that glutamate released from the photoreceptors may play a role in modulating the conductance of electrical synapses in the OPL.

OFF midget bipolar cells in the retina of the marmoset, Callithrix jacchus, express AMPA receptors.

Recent studies suggested that different types of OFF bipolar cells express specific types of ionotropic (AMPA or kainate) glutamate receptors (GluRs) at their contacts with cone pedicles. However, the question of which GluR type is expressed by which type of OFF bipolar cell in primate retina is still open. In this study, the expression of AMPA and kainate receptor subunits at the dendritic tips of flat (OFF) midget bipolar (FMB) cells was analyzed in the retina of the common marmoset, Callithrix jacchus. We used preembedding electron microscopy and double immunofluorescence with subunit‐specific antibodies. The FMB cells were labeled with antibodies against the carbohydrate epitope CD15. Cone pedicles were identified with peanut agglutinin. Immunoreactivity for the GluR1 subunit and for CD15 is preferentially located at triad‐associated flat contacts. Furthermore, the large majority of GluR1 immunoreactive puncta is localized at the dendritic tips of FMB cells. These results suggest that FMB cells express the AMPA receptor subunit GluR1. In contrast, the kainate receptor subunit GluR5 is not colocalized with the dendritic tips of FMB cells or with the GluR1 subunit. Immunoreactive puncta for the GluR1 subunit are found at all M/L‐cone pedicles but are only rarely associated with S‐cone pedicles. This is consistent with our recent findings in marmoset retina that FMB cells do not contact S‐cone pedicles. The presence of GluR5 clusters at S‐cone pedicles indicates that in primate retinas OFF bipolar cells expressing kainate receptor subunits receive some S‐cone input. J. Comp. Neurol. 502:442–454, 2007.

Bipolar cells of the ground squirrel retina.

Parallel processing of an image projected onto the retina starts at the first synapse, the cone pedicle, and each cone feeds its light signal into a minimum of eight different bipolar cell types. Hence, the morphological classification of bipolar cells is a prerequisite for analyzing retinal circuitry. Here we applied common bipolar cell markers to the cone‐dominated ground squirrel retina, studied the labeling by confocal microscopy and electron microscopy, and compared the resulting bipolar cell types with those of the mouse (rod dominated) and primate retina. Eight different cone bipolar cell types (three OFF and five ON) and one rod bipolar cell were distinguished. The major criteria for classifying the cells were their immunocytochemical identity, their dendritic branching pattern, and the shape and stratification level of their axons in the inner plexiform layer (IPL). Immunostaining with antibodies against Gγ13, a marker for ON bipolar cells, made it possible to separate OFF and ON bipolars. Recoverin‐positive OFF bipolar cells partly overlapped with ON bipolar axon terminals at the ON/OFF border of the IPL. Antibodies against HCN4 labeled the S‐cone selective (bb) bipolar cell. The calcium‐binding protein CaB5 was expressed in two OFF and two ON cone bipolar cell types, and CD15 labeled a widefield ON cone bipolar cell comparable to the DB6 in primate. J. Comp. Neurol. 519:759–774, 2011.

Cell‐type‐specific localization of protocadherin β16 at AMPA and AMPA/kainate receptor‐containing synapses in the primate retina

Protocadherins (Pcdhs) are thought to be key features of cell‐type‐specific synapse formation. Here we analyzed the expression pattern of Pcdh subunit β16 (β16) in the primate retina by applying antibodies against β16, different subunits of ionotropic glutamate receptors (GluRs), and cell‐type‐specific markers as well as by coimmunoprecipitation and Western blots. Immunocytochemical localization was analyzed by confocal microscopy and preembedding electron microscopy. In the outer plexiform layer (OPL) H1, but not H2, horizontal cells expressed β16 as revealed by the strong reduction of β16 at short‐wavelength‐sensitive cones. β16 colocalized with the GluR subunits GluR2–4 at horizontal cell dendritic tips and with GluR2–4 and GluR6/7 at the desmosome‐like junctions. At the latter, these AMPA and kainate receptor subunits were found to be clustered within single synaptic hot spots. Additionally, β16‐labeled dendritic tips of OFF cone bipolar cells appeared in triad‐associated positions at the cone pedicle base, pointing to β16 expression by OFF midget or DB3 bipolar cells. In the inner plexiform layer, β16 was localized also postsynaptically at most of the glutamatergic synapses. Overall, we provide evidence for a cell‐type‐specific localization of β16 together with GluRs at defined postsynaptic sites and a coexistence of AMPA and kainate receptors within single synaptic hot spots. This study supports the hypothesis that β16 plays an important role in the formation and/or stabilization of specific glutamatergic synapses, whereas our in vivo protein biochemical results argue against the existence of protein complexes formed by β16 and GluRs. J. Comp. Neurol. 519:467–479, 2011.

Distinctive receptive field and physiological properties of a wide-field amacrine cell in the macaque monkey retina.

At early stages of visual processing, receptive fields are typically described as subtending local regions of space and thus performing computations on a narrow spatial scale. Nevertheless, stimulation well outside of the classical receptive field can exert clear and significant effects on visual processing. Given the distances over which they occur, the retinal mechanisms responsible for these long-range effects would certainly require signal propagation via active membrane properties. Here the physiology of a wide-field amacrine cell—the wiry cell—in macaque monkey retina is explored, revealing receptive fields that represent a striking departure from the classic structure. A single wiry cell integrates signals over wide regions of retina, 5–10 times larger than the classic receptive fields of most retinal ganglion cells. Wiry cells integrate signals over space much more effectively than predicted from passive signal propagation, and spatial integration is strongly attenuated during blockade of NMDA spikes but integration is insensitive to blockade of NaV channels with TTX. Thus these cells appear well suited for contributing to the long-range interactions of visual signals that characterize many aspects of visual perception.

Broad Thorny Ganglion Cells: A Candidate for Visual Pursuit Error Signaling in the Primate Retina

Functional analyses exist only for a few of the morphologically described primate ganglion cell types, and their correlates in other mammalian species remain elusive. Here, we recorded light responses of broad thorny cells in the whole-mounted macaque retina. They showed ON-OFF-center light responses that were strongly suppressed by stimulation of the receptive field surround. Spike responses were delayed compared with parasol ganglion cells and other ON-OFF cells, including recursive bistratified ganglion cells and A1 amacrine cells. The receptive field structure was shaped by direct excitatory synaptic input and strong presynaptic and postsynaptic inhibition in both ON and OFF pathways. The cells responded strongly to dark or bright stimuli moving either in or out of the receptive field, independent of the direction of motion. However, they did not show a maintained spike response either to a uniform background or to a drifting plaid pattern. These properties could be ideally suited for guiding movements involved in visual pursuit. The functional characteristics reported here permit the first direct cross-species comparison of putative homologous ganglion cell types. Based on morphological similarities, broad thorny ganglion cells have been proposed to be homologs of rabbit local edge detector ganglion cells, but we now show that the two cells have quite distinct physiological properties. Thus, our data argue against broad thorny cells as the homologs of local edge detector cells.

Synaptic Elements for GABAergic Feed-Forward Signaling between HII Horizontal Cells and Blue Cone Bipolar Cells Are Enriched beneath Primate S-Cones

The functional roles and synaptic features of horizontal cells in the mammalian retina are still controversial. Evidence exists for feedback signaling from horizontal cells to cones and feed-forward signaling from horizontal cells to bipolar cells, but the details of the latter remain elusive. Here, immunohistochemistry and confocal microscopy were used to analyze the expression patterns of the SNARE protein syntaxin-4, the GABA receptor subunits α1 and ρ, and the cation-chloride cotransporters NKCC and KCC2 in the outer plexiform layer of primate retina. In macaque retina, as observed previously in other species, syntaxin-4 was expressed on dendrites and axon terminals of horizontal cells at cone pedicles and rod spherules. At cones, syntaxin-4 appeared densely clustered in two bands, at horizontal cell dendritic tips and at the level of desmosome-like junctions. Interestingly, in the lower band where horizontal cells may synapse directly onto bipolar cells, syntaxin-4 was highly enriched beneath short-wavelength sensitive (S) cones and colocalized with calbindin, a marker for HII horizontal cells. The enrichment at S-cones was not observed in either mouse or ground squirrel. Furthermore, high amounts of both GABA receptor and cation-chloride cotransporter subunits were found beneath primate S-cones. Finally, while syntaxin-4 was expressed by both HI and HII horizontal cell types, the intense clustering and colocalization with calbindin at S-cones indicated an enhanced expression in HII cells. Taken together, GABA receptors beneath cone pedicles, chloride transporters, and syntaxin-4 are putative constituents of a synaptic set of proteins which would be required for a GABA-mediated feed-forward pathway via horizontal cells carrying signals directly from cones to bipolar cells.