Bipolar Cells In Mouse Retina Research Articles

The Atoh1-Cre Knock-In Allele Ectopically Labels a Subpopulation of Amacrine Cells and Bipolar Cells in Mouse Retina.

The retina has diverse neuronal cell types derived from a common pool of retinal progenitors. Many molecular drivers, mostly transcription factors, have been identified to promote different cell fates. In Drosophila, atonal is required for specifying photoreceptors. In mice, there are two closely related atonal homologs, Atoh1 and Atoh7 While Atoh7 is known to promote the genesis of retinal ganglion cells, there is no study on the function of Atoh1 in retinal development. Here, we crossed Atoh1Cre/+ mice to mice carrying a Cre-dependent TdTomato reporter to track potential Atoh1-lineage neurons in retinas. We characterized a heterogeneous group of TdTomato+ retinal neurons that were detected at the postnatal stage, including glutamatergic amacrine cells, AII amacrine cells, and BC3b bipolar cells. Unexpectedly, we did not observe TdTomato+ retinal neurons in the mice with an Atoh1-FlpO knock-in allele and a Flp-dependent TdTomato reporter, suggesting Atoh1 is not expressed in the mouse retina. Consistent with these data, conditional removal of Atoh1 in the retina did not cause any observable phenotypes. Importantly, we did not detect Atoh1 expression in the retina at multiple ages using mice with Atoh1-GFP knock-in allele. Therefore, we conclude that Atoh1Cre/+ mice have ectopic Cre expression in the retina and that Atoh1 is not required for retinal development.

Center-surround interactions underlie bipolar cell motion sensitivity in the mouse retina.

Motion sensing is a critical aspect of vision. We studied the representation of motion in mouse retinal bipolar cells and found that some bipolar cells are radially direction selective, preferring the origin of small object motion trajectories. Using a glutamate sensor, we directly observed bipolar cells synaptic output and found that there are radial direction selective and non-selective bipolar cell types, the majority being selective, and that radial direction selectivity relies on properties of the center-surround receptive field. We used these bipolar cell receptive fields along with connectomics to design biophysical models of downstream cells. The models and additional experiments demonstrated that bipolar cells pass radial direction selective excitation to starburst amacrine cells, which contributes to their directional tuning. As bipolar cells provide excitation to most amacrine and ganglion cells, their radial direction selectivity may contribute to motion processing throughout the visual system.

A Systematic Evaluation of Interneuron Morphology Representations for Cell Type Discrimination

Quantitative analysis of neuronal morphologies usually begins with choosing a particular feature representation in order to make individual morphologies amenable to standard statistics tools and machine learning algorithms. Many different feature representations have been suggested in the literature, ranging from density maps to intersection profiles, but they have never been compared side by side. Here we performed a systematic comparison of various representations, measuring how well they were able to capture the difference between known morphological cell types. For our benchmarking effort, we used several curated data sets consisting of mouse retinal bipolar cells and cortical inhibitory neurons. We found that the best performing feature representations were two-dimensional density maps, two-dimensional persistence images and morphometric statistics, which continued to perform well even when neurons were only partially traced. Combining these feature representations together led to further performance increases suggesting that they captured non-redundant information. The same representations performed well in an unsupervised setting, implying that they can be suitable for dimensionality reduction or clustering.

Classification of Mouse Retinal Bipolar Cells: Type-Specific Connectivity with Special Reference to Rod-Driven AII Amacrine Pathways.

We confirmed the classification of 15 morphological types of mouse bipolar cells by serial section transmission electron microscopy and characterized each type by identifying chemical synapses and gap junctions at axon terminals. Although whether the previous type 5 cells consist of two or three types was uncertain, they are here clustered into three types based on the vertical distribution of axonal ribbons. Next, while two groups of rod bipolar (RB) cells, RB1, and RB2, were previously proposed, we clarify that a half of RB1 cells have the intermediate characteristics, suggesting that these two groups comprise a single RB type. After validation of bipolar cell types, we examined their relationship with amacrine cells then particularly with AII amacrine cells. We found a strong correlation between the number of amacrine cell synaptic contacts and the number of bipolar cell axonal ribbons. Formation of bipolar cell output at each ribbon synapse may be effectively regulated by a few nearby inhibitory inputs of amacrine cells which are chosen from among many amacrine cell types. We also found that almost all types of ON cone bipolar cells frequently have a minor group of midway ribbons along the axon passing through the OFF sublamina as well as a major group of terminal ribbons in the ON sublamina. AII amacrine cells are connected to five of six OFF bipolar cell types via conventional chemical synapses and seven of eight ON (cone) bipolar cell types via electrical synapses (gap junctions). However, the number of synapses is dependent on bipolar cell types. Type 2 cells have 69% of the total number of OFF bipolar chemical synaptic contacts with AII amacrine cells and type 6 cells have 46% of the total area of ON bipolar gap junctions with AII amacrine cells. Both type 2 and 6 cells gain the greatest access to AII amacrine cell signals also share those signals with other types of bipolar cells via networked gap junctions. These findings imply that the most sensitive scotopic signal may be conveyed to the center by ganglion cells that have the most numerous synapses with type 2 and 6 cells.

Synaptogenesis and synaptic protein localization in the postnatal development of rod bipolar cell dendrites in mouse retina.

Retinal responses to photons originate in rod photoreceptors and are transmitted to the ganglion cell output of the retina through the primary rod bipolar pathway. At the first synapse of this pathway, input from multiple rods is pooled into individual rod bipolar cells. This architecture is called convergence. Convergence serves to improve sensitivity of rod vision when photons are sparse. Establishment of convergence depends on the development of a proper complement of dendritic tips and transduction proteins in rod bipolar cells. How the dendrites of rod bipolar cells develop and contact the appropriate number of rods is unknown. To answer this question we visualized individual rod bipolar cells in mouse retina during postnatal development and quantified the number of dendritic tips, as well as the expression of transduction proteins within dendrites. Our findings show that the number of dendritic tips in rod bipolar cells increases monotonically during development. The number of tips at P21, P30, and P82 exceeds the previously reported rod convergence ratios, and the majority of these tips are proximal to a presynaptic rod release site, suggesting more rods provide input to a rod bipolar cell. We also show that dendritic transduction cascade members mGluR6 and TRPM1 appear in tips with different timelines. These finding suggest that (a) rod bipolar cell dendrites elaborate without pruning during development, (b) the convergence ratio between rods and rod bipolar cells may be higher than previously reported, and (c) mGluR6 and TRPM1 are trafficked independently during development.

Comprehensive Classification of Retinal Bipolar Neurons by Single-Cell Transcriptomics

Patterns of gene expression can be used to characterize and classify neuronal types. It is challenging, however, to generate taxonomies that fulfill the essential criteria of being comprehensive, harmonizing with conventional classification schemes, and lacking superfluous subdivisions of genuine types. To address these challenges, we used massively parallel single-cell RNA profiling and optimized computational methods on a heterogeneous class of neurons, mouse retinal bipolar cells (BCs). From a population of ∼25,000 BCs, we derived a molecular classification that identified 15 types, including all types observed previously and two novel types, one of which has anon-canonical morphology and position. We validated the classification scheme and identified dozens of novel markers using methods that match molecular expression to cell morphology. This work provides a systematic methodology for achieving comprehensive molecular classification of neurons, identifies novel neuronal types, and uncovers transcriptional differences that distinguish types within a class.

Neurotransmission plays contrasting roles in the maturation of inhibitory synapses on axons and dendrites of retinal bipolar cells

Neuronal output is modulated by inhibition onto both dendrites and axons. It is unknown whether inhibitory synapses at these two cellular compartments of an individual neuron are regulated coordinately or separately during in vivo development. Because neurotransmission influences synapse maturation and circuit development, we determined how loss of inhibition affects the expression of diverse types of inhibitory receptors on the axon and dendrites of mouse retinal bipolar cells. We found that axonal GABA but not glycine receptor expression depends on neurotransmission. Importantly, axonal and dendritic GABAA receptors comprise distinct subunit compositions that are regulated differentially by GABA release: Axonal GABAA receptors are down-regulated but dendritic receptors are up-regulated in the absence of inhibition. The homeostatic increase in GABAA receptors on bipolar cell dendrites is pathway-specific: Cone but not rod bipolar cell dendrites maintain an up-regulation of receptors in the transmission deficient mutants. Furthermore, the bipolar cell GABAA receptor alterations are a consequence of impaired vesicular GABA release from amacrine but not horizontal interneurons. Thus, inhibitory neurotransmission regulates in vivo postsynaptic maturation of inhibitory synapses with contrasting modes of action specific to synapse type and location.

LRIT3 is essential to localize TRPM1 to the dendritic tips of depolarizing bipolar cells and may play a role in cone synapse formation.

Mutations in LRIT3 lead to complete congenital stationary night blindness (cCSNB). The exact role of LRIT3 in ON-bipolar cell signaling cascade remains to be elucidated. Recently, we have characterized a novel mouse model lacking Lrit3 [no b-wave 6, (Lrit3(nob6/nob6) )], which displays similar abnormalities to patients with cCSNB with LRIT3 mutations. Here we compare the localization of components of the ON-bipolar cell signaling cascade in wild-type and Lrit3(nob6/nob6) retinal sections by immunofluorescence confocal microscopy. An anti-LRIT3 antibody was generated. Immunofluorescent staining of LRIT3 in wild-type mice revealed a specific punctate labeling in the outer plexiform layer (OPL), which was absent in Lrit3(nob6/nob6) mice. LRIT3 did not co-localize with ribeye or calbindin but co-localized with mGluR6. TRPM1 staining was severely decreased at the dendritic tips of all depolarizing bipolar cells in Lrit3(nob6/nob6) mice. mGluR6, GPR179, RGS7, RGS11 and Gβ5 immunofluorescence was absent at the dendritic tips of cone ON-bipolar cells in Lrit3(nob6/nob6) mice, while it was present at the dendritic tips of rod bipolar cells. Furthermore, peanut agglutinin (PNA) labeling was severely reduced in the OPL in Lrit3(nob6/nob6) mice. This study confirmed the localization of LRIT3 at the dendritic tips of depolarizing bipolar cells in mouse retina and demonstrated the dependence of TRPM1 localization on the presence of LRIT3. As tested components of the ON-bipolar cell signaling cascade and PNA revealed disrupted localization, an additional function of LRIT3 in cone synapse formation is suggested. These results point to a possibly different regulation of the mGluR6 signaling cascade between rod and cone ON-bipolar cells.

Ectopic synaptic ribbons in dendrites of mouse retinal ON- and OFF-bipolar cells

The ectopic distribution of synaptic ribbons in dendrites of mouse retinal bipolar cells was examined by using genetic ablation of metabotropic glutamate receptor subtype 6 (mGluR6), electron microscopy, and immunocytochemistry. Ectopic ribbons were observed in dendrites of rod and ON-cone bipolar cells in the mGluR6-deficient mouse but not in those of wild-type mice. The number of rod spherules facing the ectopic ribbons in mGluR6-deficient rod bipolar dendrites increased gradually during early growth and reached a plateau level of about 20% at 12 weeks. These ectopic ribbons were immunopositive for RIBEYE, a ribbon-specific protein, but the associated vesicles were immunonegative for synaptophysin, a synaptic-vesicle-specific protein. The presence of ectopic ribbons was correlated with an increase in the roundness of the invaginating dendrites of the rod bipolar cells. We further confirmed ectopic ribbons in dendrites of OFF-cone bipolar cells in wild-type retinas. Of the four types of OFF-cone bipolar cells (T1–T4), only the T2-type, which had a greater number of synaptic ribbons at the axon terminal and a thicker axon cylinder than the other types, had ectopic ribbons. Light-adapted experiments revealed that, in wild-type mice under enhanced-light adaptation (considered similar to the mGluR6-deficient state), the roundness in the invaginating dendrites and axon terminals of rod bipolar cells increased, but no ectopic ribbons were detected. Based on these findings and known mechanisms for neurotransmitter release and protein trafficking, the possible mechanisms underlying the ectopic ribbons are discussed on the basis of intracellular transport for the replenishment of synaptic proteins.

A Novel Connection between Rods and ON Cone Bipolar Cells Revealed by Ectopic Metabotropic Glutamate Receptor 7 (mGluR7) in mGluR6-Deficient Mouse Retinas

Since the discovery of direct chemical synapses between rod photoreceptor and OFF cone bipolar cells in mouse retinas, whether the ON cone bipolar cell also receive direct chemical input from rod has been a pending question. In finding that metabotropic glutamate receptor 7 (mGluR7) was uniquely expressed in dendrites of ON cone bipolar cells in the mGluR6-deficient mouse retina, we used this ectopic mGluR7 immunoreactivity as a specific marker for the ON cone bipolar to search for its rod connection. Here, we show that a certain type of ON cone bipolar cell forms ribbon-associated synapses not only with cones, but also rods. This finding was verified in the wild-type mouse retina by three-dimensional reconstruction of bipolar cells from serial electron micrographs. These ON cone bipolars were further identified as corresponding to type 7 of mouse bipolar cell described by Ghosh et al. (2004) and also to the green fluorescent protein (GFP)-labeled type 7 bipolars in the alpha-gustducin-GFP transgenic mouse. Our findings suggest that, in mice, rod signals bifurcate into a third ON and OFF pathway in addition to the two known routes to cone bipolar cells: (1) via rod chemical synapse --> rod bipolar --> AII amacrine --> ON and OFF cone bipolar cells; (2) via rod-cone gap junction --> cone chemical synapse --> ON and OFF cone bipolar cells; and (3) via rod chemical synapse --> ON and OFF cone bipolar cells. This third novel pathway is thought to transmit fast and moderately light-sensitive rod signals, functioning to smooth out the intensity changes at the scotopic-mesopic interface.