The retina contains distinct types of ganglion cells, which form mosaics with cells of each type at each position of the visual field. Displaced retinal ganglion cells (dRGCs) occur with cell bodies in the inner nuclear layer (INL), and regularly placed RGCs with cell bodies in the ganglion cell layer. An example of mammalian dRGCs are M1-type intrinsically photosensitive ganglion cells (ipRGCs). Little is known, however, about their relationship with regularly placed ipRGCs. We identified mouse ipRGC types M1, M2, and M4/sONɑ by immunohistochemistry and light microscopy. Reconstruction of immunolabeled mosaics from M1 and sONɑ RGCs indicated that dRGCs tiled the retina with their regular RGC partners. Multi-electrode array recordings revealed conventional receptive fields of displaced sONɑ RGCs which fit into the mosaic of their regular counterparts. An RGC distribution analysis showed type-specific dRGC patterns which followed neither the global density distribution of all RGCs nor the local densities of corresponding cell types. The displacement of RGC bodies into the INL occurs in a type-dependent manner, where dRGCs are positioned to form complete mosaics with their regular partners. Our data suggest that dRGCs and regular RGCs serve the same functional role within their corresponding population of RGCs.

In contrast to most parts of the vertebrate nervous system, ganglion cell axons in the retina typically lack myelination. In the majority of species, ganglion cell axons only become myelinated after leaving the retina to form the optic nerve. The avian retina, however, presents a remarkable exception in that ganglion cell axons are partly myelinated in the retinal nerve fibre layer. It was hypothesized that the optically detrimental properties of retinal myelination are evolutionarily offset by advantages in spike conduction velocity. Using high-resolution multielectrode array recordings, we analysed the spike conduction in the retina of various avian species in comparison to mammalian species. Indeed, mammals showed lower conduction velocities than avian species. Myelinated axons typically achieved higher conduction velocities than unmyelinated axons. Notably, some myelinated axons exhibited conduction velocities lower than those of unmyelinated axons. Anatomical analyses revealed that myelination in the nerve fibre layer was accompanied by the formation of nodes of Ranvier. The internode length was positively correlated with the axon diameter. In physiological recordings, the spatial extent of simultaneously active nodes was positively correlated with the conduction velocity. Conversely, the internode length and the activation kinetics of a node were weak predictors of conduction velocity. Overall, this study illuminates the unique features of the avian retina and offers insights into the functional requirements and evolutionary pressures of myelination affecting conduction velocity in the nervous system. KEY POINTS: Intraretinal saltatory axonal spike conduction was studied across multiple avian species using high-resolution multielectrode arrays. The highest conduction velocities were observed exclusively in saltatory axons, while the lowest were found in non-saltatory axons. However, slow myelinated axons exist, exhibiting a surprisingly large overlap in conduction velocities with unmyelinated axons. The spatial extent of a spike showed a strong positive correlation with conduction velocity. The internodal length exhibited a positive correlation with axon diameter, and the variability in internodal length was smaller within individual axons than across axons. Intraretinal axonal conduction velocities across species appear to align with their ecological niche. The maximal intraretinal spike conduction velocity observed in birds was up to four times faster than in rodents.

The topographic complexity of the mouse retina has long been underestimated. However, functional gradients exist, which reflect the non-uniform statistics of the visual environment. Horizontal cells are the first visual interneurons that shape the receptive fields of down-stream neurons. We asked whether regional specializations are present in terms of horizontal cell density distributions, morphological properties, localization of gap junction proteins, and the spatial extent of electrical coupling. These key features were asymmetrically organized along the dorsoventral axis. Dorsal cells were less densely distributed, had larger dendritic trees, and electrical coupling was more extensive than in ventral cells. The steepest change occurred at the visual horizon. Our results show that the cellular and synaptic organization of the mouse visual system are adapted to the visual environment at the earliest possible level and that horizontal cells are suited to form the substrate for the global gradient of ganglion cell receptive fields.

The mammalian retina contains many distinct types of ganglion cells, which form mosaics to evenly tile the retina with cells of each type at each position of the visual field. It is well known that displaced retinal ganglion cells (dRGCs) exist with cell bodies in the inner nuclear layer, along with regularly placed RGCs with cell bodies in the ganglion cell layer. A prominent example of dRGCs are M1-type intrinsically photosensitive ganglion cells (ipRGCs) which exist in various species including humans and non-human primates. Little is known, however, about their spatial relationship with regularly placed ipRGCs. Here, we identified mouse ipRGC types M1, M2, and M4/sONɑ by immunohistochemistry and light microscopy to anatomically investigate the distribution of displaced and regularly placed cells. Reconstruction of immunolabeled dendritic mosaics from M1 and sONɑ RGCs indicated that dRGCs tiled the retina evenly with their regularly placed RGC partners. Multi-electrode array recordings revealed conventional receptive fields of displaced sONɑ RGCs which fit into the functional mosaic of their regularly placed counterparts. We further analyzed the RGC distributions across complete retinas. The analysis of regularly placed M1 ipRGCs and ɑRGCs revealed distinct density gradients where ∼16% and ∼8% occurred as dRGCs, respectively. The density distributions of dRGCs showed type-specific patterns which followed neither the global density distribution of all ganglion cells nor the local densities of corresponding cell types. Our study shows that the displacement of ganglion cell bodies into the inner nuclear layer occurs in a type-dependent manner, where dRGCs are positioned to form complete mosaics with their regularly placed RGC partners. Our data suggest that dRGCs and regularly placed RGCs serve the same functional role within their corresponding population of ganglion cells. Significance statement We applied large-scale anatomical and electrophysiological experiments in mice to show that displaced intrinsically photosensitive retinal ganglion cells (ipRGCs) complete the mosaics of their regularly placed counterparts with their dendritic trees and receptive fields. Therefore, displaced ipRGCs likely serve the same functional role as corresponding regularly placed cells. The density distributions of displaced ipRGCs showed distinct, type-specific patterns. Interestingly, they followed neither the global density distribution of all ganglion cells nor the local densities of corresponding cell types.

From mouse to primate, there is a striking discontinuity in our current understanding of the neural coding of motion direction. In non-primate mammals, directionally selective cell types and circuits are a signature feature of the retina, situated at the earliest stage of the visual process. In primates, by contrast, direction selectivity is a hallmark of motion processing areas in visual cortex, but has not been found in the retina, despite significant effort. Here we combined functional recordings of light-evoked responses and connectomic reconstruction to identify diverse direction-selective cell types in the macaque monkey retina with distinctive physiological properties and synaptic motifs. This circuitry includes an ON-OFF ganglion cell type, a spiking, ON-OFF polyaxonal amacrine cell and the starburst amacrine cell, all of which show direction selectivity. Moreover, we discovered that macaque starburst cells possess a strong, non-GABAergic, antagonistic surround mediated by input from excitatory bipolar cells that is critical for the generation of radial motion sensitivity in these cells. Our findings open a door to investigation of a precortical circuitry that computes motion direction in the primate visual system.

In primates, broad thorny retinal ganglion cells are highly sensitive to small, moving stimuli. They have tortuous, fine dendrites with many short, spine-like branches that occupy three contiguous strata in the middle of the inner plexiform layer. The neural circuits that generate their responses to moving stimuli are not well-understood, and that was the goal of this study. A connectome from central macaque retina was generated by serial block-face scanning electron microscopy, a broad thorny cell was reconstructed, and its synaptic inputs were analyzed. It received fewer than 2% of its inputs from both ON and OFF types of bipolar cells; the vast majority of its inputs were from amacrine cells. The presynaptic amacrine cells were reconstructed, and seven types were identified based on their characteristic morphology. Two types of narrow-field cells, knotty bistratified Type 1 and wavy multistratified Type 2, were identified. Two types of medium-field amacrine cells, ON starburst and spiny, were also presynaptic to the broad thorny cell. Three types of wide-field amacrine cells, wiry Type 2, stellate wavy, and semilunar Type 2, also made synapses onto the broad thorny cell. Physiological experiments using a macaque retinal preparation in vitro confirmed that broad thorny cells received robust excitatory input from both the ON and the OFF pathways. Given the paucity of bipolar cell inputs, it is likely that amacrine cells provided much of the excitatory input, in addition to inhibitory input.

Electrical coupling has been reported to occur only between homotypic retinal ganglion cells, in line with the concept of parallel processing in the early visual system. Here, however, we show reciprocal correlated firing between heterotypic ganglion cells in multielectrode array recordings during light stimulation in retinas of adult guinea pigs of either sex. Heterotypic coupling was further confirmed via tracer spread after intracellular injections of single cells with neurobiotin. Both electrically coupled cell types were sustained ON center ganglion cells but showed distinct light response properties and receptive field sizes. We identified one of the involved cell types as sustained ON α-ganglion cells. The presence of electrical coupling between heterotypic ganglion cells introduces a network motif in which the signals of distinct ganglion cell types are partially mixed at the output stage of the retina.SIGNIFICANCE STATEMENT The visual information is split into parallel pathways, before it is sent to the brain via the output neurons of the retina, the ganglion cells. Ganglion cells can form electrical synapses between dendrites of neighboring cells in support of lateral information exchange. To date, ganglion-to-ganglion cell coupling is thought to occur only between cells of the same type. Here, however, we show that electrical coupling between different types of ganglion cells exists in the mammalian retina. We provide functional and anatomical evidence that two different types of ganglion cells share information via electrical coupling. This new network motif extends the impact of the heavily studied coding benefits of homotypic coupling to heterotypic coupling across parallel neuronal pathways.

In the mammalian retina, horizontal cells receive glutamatergic inputs from many rod and cone photoreceptors and return feedback signals to them, thereby changing photoreceptor glutamate release in a light-dependent manner. Horizontal cells also provide feedforward signals to bipolar cells. It is unclear, however, how horizontal cell signals also affect the temporal, spatial, and contrast tuning in retinal output neurons, the ganglion cells. To study this, we generated a genetically modified mouse line in which we eliminated the light dependency of feedback by deleting glutamate receptors from mouse horizontal cells. This genetic modification allowed us to investigate the impact of horizontal cells on ganglion cell signaling independent of the actual mode of feedback in the outer retina and without pharmacological manipulation of signal transmission. In control and genetically modified mice (both sexes), we recorded the light responses of transient OFF-α retinal ganglion cells in the intact retina. Excitatory postsynaptic currents (EPSCs) were reduced and the cells were tuned to lower temporal frequencies and higher contrasts, presumably because photoreceptor output was attenuated. Moreover, receptive fields of recorded cells showed a significantly altered surround structure. Our data thus suggest that horizontal cells are responsible for adjusting the dynamic range of retinal ganglion cells and, together with amacrine cells, contribute to the center/surround organization of ganglion cell receptive fields in the mouse. SIGNIFICANCE STATEMENT Horizontal cells represent a major neuronal class in the mammalian retina and provide lateral feedback and feedforward signals to photoreceptors and bipolar cells, respectively. The mode of signal transmission remains controversial and, moreover, the contribution of horizontal cells to visual processing is still elusive. To address the question of how horizontal cells affect retinal output signals, we recorded the light responses of transient OFF-α retinal ganglion cells in a newly generated mouse line. In this mouse line, horizontal cell signals were no longer modulated by light. With light response recordings, we show that horizontal cells increase the dynamic range of retinal ganglion cells for contrast and temporal changes and contribute to the center/surround organization of their receptive fields.

Retinal OFF bipolar cells show distinct connectivity patterns with photoreceptors in the wild-type mouse retina. Some types are cone-specific while others penetrate further through the outer plexiform layer (OPL) to contact rods in addition to cones. To explore dendritic stratification of OFF bipolar cells in the absence of rods, we made use of the ‘cone-full’ Nrl-/- mouse retina in which all photoreceptor precursor cells commit to a cone fate including those which would have become rods in wild-type retinas. The dendritic distribution of OFF bipolar cell types was investigated by confocal and electron microscopic imaging of immunolabeled tissue sections. The cells’ dendrites formed basal contacts with cone terminals and expressed the corresponding glutamate receptor subunits at those sites, indicating putative synapses. All of the four analyzed cell populations showed distinctive patterns of vertical dendritic invasion through the OPL. This disparate behavior of dendritic extension in an environment containing only cone terminals demonstrates type-dependent specificity for dendritic outgrowth in OFF bipolar cells: rod terminals are not required for inducing dendritic extension into distal areas of the OPL.

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.

1 1 2 3 4 5 6 Distinctive receptive field and physiological properties of a wide-field amacrine cell in the macaque 7 monkey retina 8 9 10 Michael B. Manookin, Christian Puller, Fred Rieke, Jay Neitz, Maureen Neitz 11 Department of Ophthalmology, Physiology and Biophysics Department, HHMI, University of 12 Washington, Seattle, WA 98109 13 14 Corresponding authors: Michael B. Manookin (manookin@uw.edu), Maureen Neitz (mneitz@uw.edu) 15 16 ACKNOWLEDGEMENTS 17 We are grateful to Martin Greschner for detailed comments on an earlier version of the manuscript, Mark 18 Cafaro, Paul Newman, Shellee Cunnington, Jing Huang, and Dan Possin for technical assistance, and 19 Juan Angueyra, Raunak Sinha, and the Tissue Distribution Program of the Washington National Primate 20 Research Center (supported through NIH grant P51 OD010425) for providing primate tissue. This work 21 was supported by the Helen Hay Whitney Foundation, German Research Foundation (DFG PU 469/2-1), 22 NIH/NEI (R01 EY09303, R01 EY11850, F32 EY024507, P30 EY01730), Research to Prevent Blindness, 23 and Howard Hughes Medical Institute. 24 25 26 Articles in PresS. J Neurophysiol (July 1, 2015). doi:10.1152/jn.00484.2015

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 Na V 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.

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.

The distribution of the soluble NSF-attachment protein receptor protein syntaxin-4 and the Na-K-Cl cotransporter (NKCC) were investigated in the outer plexiform layer of human retina using immunohistochemistry. Both proteins, which are proposed to be components of a gamma-aminobutyric acid mediated feed-forward circuit from horizontal cells directly to bipolar cells, were enriched beneath S-cones. The expression pattern of syntaxin-4 was further analyzed in baboon and marmoset to determine if the synaptic specialization is common to primates. Syntaxin-4 was enriched beneath S-cones in both species, which together with the human results indicates that this specialization may have evolved for the purpose of mediating unique color vision capacities that are exclusive to primates.