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An organizational feature of neural circuits is the specificity of synaptic connections. A striking example is the direction-selective (DS) circuit of the retina. There are multiple subtypes of DS retinal ganglion cells (DSGCs) that prefer motion along one of four preferred directions. This computation is mediated by selective wiring of a single inhibitory interneuron, the starburst amacrine cell (SAC), with each DSGC subtype preferentially receiving input from a subset of SAC processes. We hypothesize that the molecular basis of this wiring is mediated in part by unique expression profiles of DSGC subtypes. To test this, we first performed paired recordings from isolated mouse retinas of both sexes to determine that postnatal day 10 (P10) represents the age at which asymmetric synapses form. Second, we performed RNA sequencing and differential expression analysis on isolated P10 ON-OFF DSGCs tuned for either nasal or ventral motion and identified candidates which may promote direction-specific wiring. We then used a conditional knock-out strategy to test the role of one candidate, the secreted synaptic organizer cerebellin-4 (Cbln4), in the development of DS tuning. Using two-photon calcium imaging, we observed a small deficit in directional tuning among ventral-preferring DSGCs lacking Cbln4, though whole-cell voltage-clamp recordings did not identify a significant change in inhibitory inputs. This suggests that Cbln4 does not function primarily via a cell-autonomous mechanism to instruct wiring of DS circuits. Nevertheless, our transcriptomic analysis identified unique candidate factors for gaining insights into the molecular mechanisms that instruct wiring specificity in the DS circuit.

Retinal prosthetics are one of the leading therapeutic strategies to restore lost vision in patients with retinitis pigmentosa and age-related macular degeneration. Much work has described patterns of spiking in retinal ganglion cells (RGCs) in response to electrical stimulation, but less work has examined the underlying retinal circuitry that is activated by electrical stimulation to drive these responses. Surprisingly, little is known about the role of inhibition in generating electrical responses or how inhibition might be altered during degeneration. Using whole-cell voltage-clamp recordings during subretinal electrical stimulation in the rd10 and wild-type (wt) retina, we found electrically evoked synaptic inputs differed between ON and OFF RGC populations, with ON cells receiving mostly excitation and OFF cells receiving mostly inhibition and very little excitation. We found that the inhibition of OFF bipolar cells limits excitation in OFF RGCs, and a majority of both pre- and postsynaptic inhibition in the OFF pathway arises from glycinergic amacrine cells, and the stimulation of the ON pathway contributes to inhibitory inputs to the RGC. We also show that this presynaptic inhibition in the OFF pathway is greater in the rd10 retina, compared with that in the wt retina.

The retina is exquisitely patterned, with neuronal somata positioned at regular intervals to completely sample the visual field. Here, we show that phosphatase and tensin homolog (Pten) controls starburst amacrine cell spacing by modulating vesicular trafficking of cell adhesion molecules and Wnt proteins. Single-cell transcriptomics and double-mutant analyses revealed that Pten and Down syndrome cell adhesion molecule Dscam) are co-expressed and function additively to pattern starburst amacrine cell mosaics. Mechanistically, Pten loss accelerates the endocytic trafficking of DSCAM, FAT3, and MEGF10 off the cell membrane and into endocytic vesicles in amacrine cells. Accordingly, the vesicular proteome, a molecular signature of the cell of origin, is enriched in exocytosis, vesicle-mediated transport, and receptor internalization proteins in Pten conditional knockout (PtencKO) retinas. Wnt signaling molecules are also enriched in PtencKO retinal vesicles, and the genetic or pharmacological disruption of Wnt signaling phenocopies amacrine cell patterning defects. Pten thus controls vesicular trafficking of cell adhesion and signaling molecules to establish retinal amacrine cell mosaics.

Gap junctions are channels that allow for direct transmission of electrical signals between cells. However, the ability of one cell to be impacted or controlled by other cells through gap junctions remains unclear. In this study, heterocellular coupling between ON α retinal ganglion cells (α-RGCs) and displaced amacrine cells (ACs) in the mouse retina was used as a model. The impact of the extent of coupling of interconnected ACs on the synchronized firing between coupled ON α-RGC-AC pair was investigated using the dopamine 1 receptor (D1R) antagonist—SCH23390 and agonist—SKF38393. It was observed that the synchronized firing between the ON α-RGC-ACs pairs was increased by the D1R antagonist SCH23390, whereas it was eradicated by the agonist SKF38393. Subsequently, the signaling drive was investigated by infecting coupled ON α-RGC-AC pairs with the channelrhodopsin-2(ChR2) mutation L132C engineered to enhance light sensitivities. The results demonstrated that the spikes of ON α-RGCs (without ChR2) could be triggered by ACs (with ChR2) through the gap junction, and vice versa. Furthermore, it was observed that ON α-RGCs stimulated with 3–10 Hz currents by whole cell patch could elicit synchronous spikes in the coupled ACs, and vice versa. This provided direct evidence that the firing of one cell could be influenced by another cell through gap junctions. However, this phenomenon was not observed between OFF α-RGC pairs. The study implied that the synchronized firing between ON α-RGC-AC pairs could potentially be affected by the coupling of interconnected ACs. Additionally, one cell type could selectively control the firing of another cell type, thereby forcefully transmitting information. The key role of gap junctions in synchronizing firing and driving cells between α-RGCs and coupled ACs in the mouse retina was highlighted. NEW & NOTEWORTHY: This study investigates the role of gap junctions in transmitting electrical signals between cells and their potential for cell control. Using ON α retinal ganglion cells (α-RGCs) and amacrine cells (ACs) in the mouse retina, the researchers find that the extent of coupling between ACs affects synchronized firing. Bidirectional signaling occurs between ACs and ON α-RGCs through gap junctions. [ABSTRACT FROM AUTHOR]

The basic plan of the retina is conserved across vertebrates, yet species differ profoundly in their visual needs1. Retinal cell types may have evolved to accommodate these varied needs, but this has not been systematically studied. Here we generated and integrated single-cell transcriptomic atlases of the retina from 17 species: humans, two non-human primates, four rodents, three ungulates, opossum, ferret, tree shrew, a bird, a reptile, a teleost fish and a lamprey. We found high molecular conservation of the six retinal cell classes (photoreceptors, horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells (RGCs) and Müller glia), with transcriptomic variation across species related to evolutionary distance. Major subclasses were also conserved, whereas variation among cell types within classes or subclasses was more pronounced. However, an integrative analysis revealed that numerous cell types are shared across species, based on conserved gene expression programmes that are likely to trace back to an early ancestral vertebrate. The degree of variation among cell types increased from the outer retina (photoreceptors) to the inner retina (RGCs), suggesting that evolution acts preferentially to shape the retinal output. Finally, we identified rodent orthologues of midget RGCs, which comprise more than 80% of RGCs in the human retina, subserve high-acuity vision, and were previously believed to be restricted to primates2. By contrast, the mouse orthologues have large receptive fields and comprise around 2% of mouse RGCs. Projections of both primate and mouse orthologous types are overrepresented in the thalamus, which supplies the primary visual cortex. We suggest that midget RGCs are not primate innovations, but are descendants of evolutionarily ancient types that decreased in size and increased in number as primates evolved, thereby facilitating high visual acuity and increased cortical processing of visual information.

Psychedelics like LSD (Lysergic acid diethylamide) and psilocybin are known to modulate perceptual modalities due to the activation of mostly serotonin receptors in specific cortical (e.g., visual cortex) and subcortical (e.g., thalamus) regions of the brain. In the visual domain, these psychedelic modulations often result in peculiar disturbances of viewed objects and light and sometimes even in hallucinations of non-existent environments, objects, and creatures. Although the underlying processes are poorly understood, research conducted over the past twenty years on the subjective experience of psychedelics details theories that attempt to explain these perceptual alterations due to a disruption of communication between cortical and subcortical regions. However, rare medical conditions in the visual system like Charles Bonnet syndrome that cause perceptual distortions may shed new light on the additional importance of the retinofugal pathway in psychedelic subjective experiences. Interneurons in the retina called amacrine cells could be the first site of visual psychedelic modulation and aid in disrupting the hierarchical structure of how humans perceive visual information. This paper presents an understanding of how the retinofugal pathway communicates and modulates visual information in psychedelic and clinical conditions. Therefore, we elucidate a new theory of psychedelic modulation in the retinofugal pathway. [ABSTRACT FROM AUTHOR]

The precise specification of cellular fate is thought to ensure the production of the correct number of neurons within a population. Programmed cell death may be an additional mechanism controlling cell number, believed to refine the proper ratio of pre- to post-synaptic neurons for a given species. Here, we consider the size of three different neuronal populations in the rod pathway of the mouse retina: rod photoreceptors, rod bipolar cells, and AII amacrine cells. Across a collection of 28 different strains of mice, large variation in the numbers of all three cell types is present. The variation in their numbers is not correlated, so that the ratio of rods to rod bipolar cells, as well as rod bipolar cells to AII amacrine cells, varies as well. Establishing connectivity between such variable pre- and post-synaptic populations relies upon plasticity that modulates process outgrowth and morphological differentiation, which we explore experimentally for both rod bipolar and AII amacrine cells in a mouse retina with elevated numbers of each cell type. While both rod bipolar dendritic and axonal arbors, along with AII lobular arbors, modulate their areal size in relation to local homotypic cell densities, the dendritic appendages of the AII amacrine cells do not. Rather, these processes exhibit a different form of plasticity, regulating the branching density of their overlapping arbors. Each form of plasticity should ensure uniformity in retinal coverage in the presence of the independent specification of afferent and target cell number.

Background: Parkinson's disease (PD) patients experience visual symptoms and retinal degeneration. Studies using optical coherence tomography (OCT) have shown reduced thickness of the retina in PD, also a key characteristic of glaucoma. Objective: To identify the presence and pattern of retinal changes in de novo, treatment-naive PD patients compared to healthy controls (HC) and early primary open angle glaucoma (POAG) patients. Methods: Macular OCT data (10×10 mm) were collected from HC, PD, and early POAG patients, at the University Medical Center Groningen. Bayesian informative hypotheses statistical analyses were carried out comparing HC, PD-, and POAG patients, within each retinal cell layer. Results: In total 100 HC, 121 PD, and 78 POAG patients were included. We showed significant reduced thickness of the inner plexiform layer and retinal pigment epithelium in PD compared to HC. POAG patients presented with a significantly thinner retinal nerve fiber layer, ganglion cell layer, inner plexiform layer, outer plexiform layer, and outer photoreceptor and subretinal virtual space compared to PD. Only the outer segment layer and retinal pigment epithelium were significantly thinner in PD compared to POAG. Conclusions: De novo PD patients show reduced thickness of the retina compared to HC, especially of the inner plexiform layer, which differs significantly from POAG, showing a more extensive and widespread pattern of reduced thickness across layers. OCT is a useful tool to detect retinal changes in de novo PD, but its specificity versus other neurodegenerative disorders has to be established. [ABSTRACT FROM AUTHOR]

The microanatomy of the donkey eye is important to understand because pathological disorders affecting them are relatively common. The current study aimed to document the cellular components of donkey's retinae using light and electron microscopic studies. Ten donkey retinae were dissected and processed for semi-thin sections and electron microscopic studies. The photoreceptor layer was made up of the outer and inner segments of rods and cones. The outer segments were filled with invaginations of cell membranes that form stacks of membranous disks. Shed discs of photoreceptor outer segments could be seen in the photoreceptor layer as well as near the Müller cells. The inner segments of cones were conical in shape, while those of rods were slim rod-shaped. Both were filled with long thin mitochondria and free ribosomes. Three rows of photoreceptor cell nuclei made up the outer nuclear layer. The rod nuclei had more electron-dense chromatin than those of the cones. There were two rows of cell nuclei in the inner nuclear layer that represent the following four cell classes: horizontal cells, bipolar cells, amacrine cells, and Müller cells. Bipolar cells constitute the bulk of the inner nuclear layer. They were elongated in shape and had thick branched dendrites. Amacrine cells were in the inner face of the INL. It could be observed within IPL and known as displaced amacrine cells. Muller glial cells were irregular elongated in shape with many cytoplasmic processes. They were distributed in the INL among the bipolar cells. Müller cells were observed in the inner plexiform layer, the ganglion cell layer, and the nerve fiber layer. In conclusion, this study characterized the detailed cytological organization and ultrastructure of the healthy donkey retina. which maintains the fundamental laminar architecture characteristic of other mammalian retinas, and consists of 10 distinguishable layers. When compared to previously described retinal morphologies in domestic species, some distinctive characters were observed in donkey retinal cells. [ABSTRACT FROM AUTHOR]

We recently identified PKN1 as a developmentally active gatekeeper of the transcription factor neuronal differentiation-2 (NeuroD2) in several brain areas. Since NeuroD2 plays an important role in amacrine cell (AC) and retinal ganglion cell (RGC) type formation, we aimed to study the expression of NeuroD2 in the postnatal retina of WT and Pkn1−/− animals, with a particular focus on these two cell types. We show that PKN1 is broadly expressed in the retina and that the gross retinal structure is not different between both genotypes. Postnatal retinal NeuroD2 levels were elevated upon Pkn1 knockout, with Pkn1−/− retinae showing more NeuroD2+ cells in the lower portion of the inner nuclear layer. Accordingly, immunohistochemical analysis revealed an increased amount of AC in postnatal and adult Pkn1−/− retinae. There were no differences in horizontal cell, bipolar cell, glial cell and RGC numbers, nor defective axon guidance to the optic chiasm or tract upon Pkn1 knockout. Interestingly, we did, however, see a specific reduction in SMI-32+ α-RGC in Pkn1−/− retinae. These results suggest that PKN1 is important for retinal cell type formation and validate PKN1 for future studies focusing on AC and α-RGC specification and development. [ABSTRACT FROM AUTHOR]

Midget and parasol ganglion cells (GCs) represent the major output channels from the primate eye to the brain. On-type midget and parasol GCs exhibit a higher background spike rate and thus can respond more linearly to contrast changes than their Off-type counterparts. Here, we show that a calcium-permeable AMPA receptor (CP-AMPAR) antagonist blocks background spiking and sustained light-evoked firing in On-type GCs while preserving transient light responses. These effects are selective for On-GCs and are occluded by a gap-junction blocker suggesting involvement of AII amacrine cells (AII-ACs). Direct recordings from AII-ACs, cobalt uptake experiments, and analyses of transcriptomic data confirm that CP-AMPARs are expressed by primate AII-ACs. Overall, our data demonstrate that under some background light levels, CP-AMPARs at the rod bipolar to AII-AC synapse drive sustained signaling in On-type GCs and thus contribute to the more linear contrast signaling of the primate On- versus Off-pathway.

Rbfox1 is a multifunctional RNA-binding protein that regulates alternative splicing, transcription, mRNA stability, and translation. Rbfox1 is an important regulator of gene networks involved in neurogenesis and neuronal function. Disruption of Rbfox function has been associated with several neurodevelopmental and neuropsychiatric disorders. We have shown earlier that Rbfox1 is expressed in retinal ganglion and amacrine cells (ACs) and that its down-regulation in adult mouse retinas leads to deficiency of depth perception. In the present study, we used several markers of ACs, including gamma-aminobutyric acid (GABA), choline acetyltransferase (ChAT), neuropeptide Y (NPY), glycine transporter (GlyT1), and vesicular glutamate transporter 3 (VGlut3) to identify types of ACs that express Rbfox1. Expression of Rbfox1 was observed predominantly in GABAergic ACs located in the inner nuclear layer (INL) and ganglion cell layer (GCL). All GABAergic/cholinergic starburst ACs and virtually all NPY-positive GABAergic ACs were also Rbfox1-positive. Among glycinergic ACs, a sparse population of Rbfox1/VGlut3-positive cells was identified, indicating that Rbfox1 is expressed in a very small population of glycinergic ACs. These data contribute to our understanding about molecular differences between various types of amacrine cells and the cell-specific gene networks regulated by Rbfox1.

Background: Loss of substantia nigra dopaminergic cells and alpha-synuclein (α-syn)-rich intraneuronal deposits within the central nervous system are key hallmarks of Parkinson's disease (PD). Levodopa (L-DOPA) is the current gold-standard treatment for PD. This study aimed to evaluate in vivo retinal changes in a transgenic PD model of α-syn overexpression and the effect of acute levodopa (L-DOPA) treatment. Methods: Anaesthetised 6-month-old mice expressing human A53T alpha-synuclein (HOM) and wildtype (WT) control littermates were intraperitoneally given 20 mg/kg L-DOPA (50 mg levodopa, 2.5 mg benserazide) or vehicle saline (n = 11–18 per group). In vivo retinal function (dark-adapted full-field ERG) and structure (optical coherence tomography, OCT) were recorded before and after drug treatment for 30 min. Ex vivo immunohistochemistry (IHC) on flat-mounted retina was conducted to assess tyrosine hydroxylase (TH) positive cell counts (n = 7–8 per group). Results: We found that photoreceptor (a-wave) and bipolar cell (b-wave) ERG responses (p < 0.01) in A53T HOM mice treated with L-DOPA grew in amplitude more (47 ± 9%) than WT mice (16 ± 9%) treated with L-DOPA, which was similar to the vehicle group (A53T HOM 25 ± 9%; WT 19 ± 7%). While outer retinal thinning (outer nuclear layer, ONL, and outer plexiform layer, OPL) was confirmed in A53T HOM mice (p < 0.01), L-DOPA did not have an ameliorative effect on retinal layer thickness. These findings were observed in the absence of changes to the number of TH-positive amacrine cells across experiment groups. Acute L-DOPA treatment transiently improves visual dysfunction caused by abnormal alpha-synuclein accumulation. Conclusions: These findings deepen our understanding of dopamine and alpha-synuclein interactions in the retina and provide a high-throughput preclinical framework, primed for translation, through which novel therapeutic compounds can be objectively screened and assessed for fast-tracking PD drug discovery. [ABSTRACT FROM AUTHOR]

Substance P (SP), a neuroprotective peptidergic neurotransmitter, is known to have immunoreactivity (IR) localized to amacrine and/or ganglion cells in a variety of species' retinas, but it has not yet been studied in the mouse retina. Thus, we investigated the distribution and synaptic organization of SP-IR by confocal and electron microscopy immunocytochemistry in the mouse retina. SP-IR was distributed in the inner nuclear layer (INL), inner plexiform layer (IPL), and ganglion cell layer (GCL). Most of the SP-IR somas belonged to amacrine cells (2.5% of all) in the INL and their processes stratified into the S1, S3, and S5 layers of the IPL, with the most intense band in the S5 layer. Some SP-IR somas can also be observed in the GCL, which were identified as displaced amacrine cells (82%, 1269/1550) and ganglion cells (18%, 281/1550) by antibodies against AP2α and RBPMS, respectively. Such SP-IR ganglion cells (1.2% of all RGCs) can be further divided into 3 subgroups expressing SP/α-Synuclein (α-Syn), SP/GAD67, and/or SP/GAD67/α-Syn. Possible physiological and pathological roles of these ganglion cells are discussed. Further, electron microscopy evidence demonstrates that SP-IR amacrine cells receive major inputs from other SP-IR amacrine cell processes (146/242 inputs) and output mostly to SP-negative amacrine cell processes (291/673 outputs), suggesting series inhibition among amacrine cells. These results reveal for the first time an explicit distribution, novel ganglion cell features, and synaptic organization of SP-IR in the mouse retina, which is important for the future use of mouse models to study the roles of SP in healthy and diseased (including Parkinson's disease) retinal states. [ABSTRACT FROM AUTHOR]

Amacrine cells of the retina are conspicuously variable in their morphologies, their population demographics, and their ensuing functions. Vesicular glutamate transporter 3 (VGluT3) amacrine cells are a recently characterized type of amacrine cell exhibiting local dendritic autonomy. The present analysis has examined three features of this VGluT3 population, including their density, local distribution, and dendritic spread, to discern the extent to which these are interrelated, using male and female mice. We first demonstrate that Bax-mediated cell death transforms the mosaic of VGluT3 cells from a random distribution into a regular mosaic. We subsequently examine the relationship between cell density and mosaic regularity across recombinant inbred strains of mice, finding that, although both traits vary across the strains, they exhibit minimal covariation. Other genetic determinants must therefore contribute independently to final cell number and to mosaic order. Using a conditional KO approach, we further demonstrate that Bax acts via the bipolar cell population, rather than cell-intrinsically, to control VGluT3 cell number. Finally, we consider the relationship between the dendritic arbors of single VGluT3 cells and the distribution of their homotypic neighbors. Dendritic field area was found to be independent of Voronoi domain area, while dendritic coverage of single cells was not conserved, simply increasing with the size of the dendritic field. Bax-KO retinas exhibited a threefold increase in dendritic coverage. Each cell, however, contributed less dendrites at each depth within the plexus, intermingling their processes with those of neighboring cells to approximate a constant volumetric density, yielding a uniformity in process coverage across the population.SIGNIFICANCE STATEMENT Different types of retinal neuron spread their processes across the surface of the retina to achieve a degree of dendritic coverage that is characteristic of each type. Many of these types achieve a constant coverage by varying their dendritic field area inversely with the local density of like-type neighbors. Here we report a population of retinal amacrine cells that do not develop dendritic arbors in relation to the spatial positioning of such homotypic neighbors; rather, this cell type modulates the extent of its dendritic branching when faced with a variable number of overlapping dendritic fields to approximate a uniformity in dendritic density across the retina.

Rbfox1 is a multifunctional RNA binding protein that regulates alternative splicing, transcription, mRNA stability and translation. Its roles in neurogenesis and neuronal functions are well established. Recent studies also implicate Rbfox1 in the regulation of gene networks that support cell survival during stress. We have earlier characterized the expression of Rbfox1 in amacrine and retinal ganglion cells (RGCs) and showed that deletion of Rbfox1 in adult animals results in depth perception deficiency. The current study investigates the effect of Rbfox1 downregulation on survival of RGCs injured by optic nerve crush (ONC). Seven days after ONC, animals sustained severe degeneration of RGC axons in the optic nerve and significant loss of RGC somas. Semi-quantitative grading of optic nerve damage in control + ONC, control + tamoxifen + ONC, and Rbfox1 -/- + ONC groups ranged from 4.6 to 4.8 on a scale of 1 (normal; no degenerated axons were noted) to 5 (total degeneration; all axons showed degenerated organelles, axonal content, and myelin sheath), indicating a severe degeneration. Among these three ONC groups, no statistical significance was observed when any two groups were compared. The number of RGC somas were quantitatively analyzed in superior, inferior, nasal and temporal retinal quadrants at 0.5, 1, and 1.5 mm from the center of the optic disc. The average RGC densities (cells/mm2) were: control 6,438 ± 1,203; control + ONC 2,779 ± 573; control + tamoxifen 6,163 ± 861; control + tamoxifen + ONC 2,573 ± 555; Rbfox1 -/- 6,437 ± 893; and Rbfox1 -/- + ONC 2,537 ± 526. The RGC loss in control + ONC, control + tamoxifen + ONC and Rbfox1 -/- + ONC was 57% (P = 1.44954E-42), 58% (P = 1.37543E-57) and 61% (P = 5.552E-59) compared to RGC numbers in the relevant uninjured groups, respectively. No statistically significant difference was observed between any two groups of uninjured animals or between any two ONC groups. Our data indicate that Rbfox1-mediated pathways have no effect on survival of RGCs injured by ONC.

Adapting between scotopic and photopic illumination involves switching the routing of retinal signals between rod and cone-dominated circuits. In the daytime, cone signals pass through parallel On and Off cone bipolar cells (CBCs), that are sensitive to increments and decrements in luminance, respectively. At night, rod signals are routed into these cone-pathways via a key glycinergic interneuron, the AII amacrine cell (AII-AC). AII-ACs also provide On-pathway-driven crossover inhibition to Off-CBCs under photopic conditions. In primates, it is not known whether all Off-bipolar cell types receive functional inputs from AII-ACs. Here, we show that select Off-CBC types receive significantly higher levels of On-pathway-driven glycinergic input than others. The rise and decay kinetics of the glycinergic events are consistent with involvement of the α1 glycine receptor (GlyR) subunit, a result supported by a higher level of GLRA1 transcript in these cells. The Off-bipolar types that receive glycinergic input have sustained physiological properties and include the flat midget bipolar (FMB) cells, which provide excitatory input to the Off-midget ganglion cells (GCs; parvocellular pathway). Our results suggest that only a subset of Off-bipolar cells have the requisite receptors to respond to AII-AC input. Taken together with results in mouse retina, our findings suggest a conserved motif whereby signal output from AII-ACs is preferentially routed into sustained Off-bipolar signaling pathways.

α‐Synuclein (α‐Syn) is enriched in presynaptic terminals of the central nervous system including the retina and plays a role in the synaptic vesicle cycle and synaptic transmission. Abnormal aggregation of α‐Syn is considered to be the main component of the Lewy bodies that are the pathological hallmarks of Parkinson's disease. Although expression pattern of α‐Syn has been described in the retinas, its precise cellular and subcellular locations are poorly understood. We investigated the precise expression of α‐Syn using light microscopy (LM) and electron microscopy (EM) with antibodies against α‐Syn in the mouse retina. We found that the majority of α‐Syn immunoreactivity (IR) is located in GABAergic, glycinergic, and dopaminergic amacrine cells, and their processes often make a direct synapse to other labeled or unlabeled amacrine profiles, bipolar cell terminals, or ganglion cell dendrites. Further, our LM and immuno‐EM results confirm the absence of α‐Syn in excitatory photoreceptors, bipolar cell bodies, and their ribbon synapses, providing evidence, for the first time, that ribbon synapses do not express α‐Syn. Additionally, α‐Syn IR is located in the ganglion cells, some of which are intrinsically photosensitive retinal ganglion cells. These results reveal a previously unappreciated inhibitory synapse‐specific expression pattern of α‐Syn in the retina, suggesting that α‐Syn may play a distinct role in the modulation and integration of inhibitory synaptic transmission in the retina. [ABSTRACT FROM AUTHOR]

The visual system of sighted animals plays a critical role in providing information about the environment, including motion details necessary for survival. Over the past few years, numerous studies have explored the mechanism of motion direction detection in the visual system for binary images, including the Hassenstein–Reichardt model (HRC model) and the HRC-based artificial visual system (AVS). In this paper, we introduced a contrast-response system based on previous research on amacrine cells in the visual system of Drosophila and other species. We combined this system with the HRC-based AVS to construct a motion-direction-detection system for gray-scale images. Our experiments verified the effectiveness of our model in detecting the motion direction in gray-scale images, achieving at least 99% accuracy in all experiments and a remarkable 100% accuracy in several circumstances. Furthermore, we developed two convolutional neural networks (CNNs) for comparison to demonstrate the practicality of our model. [ABSTRACT FROM AUTHOR]

The various types of retinal neurons are each positioned at their respective depths within the retina where they are believed to be assembled as orderly mosaics, in which like-type neurons minimize proximity to one another. Two common statistical analyses for assessing the spatial properties of retinal mosaics include the nearest neighbor analysis, from which an index of their "regularity" is commonly calculated, and the density recovery profile derived from autocorrelation analysis, revealing the presence of an exclusion zone indicative of anti-clustering. While each of the spatial statistics derived from these analyses, the regularity index and the effective radius, can be useful in characterizing such properties of orderly retinal mosaics, they are rarely sufficient for conveying the natural variation in the self-spacing behavior of different types of retinal neurons and the extent to which that behavior generates uniform intercellular spacing across the mosaic. We consider the strengths and limitations of these and other spatial statistical analyses for assessing the patterning in retinal mosaics, highlighting a number of misconceptions and their frequent misuse. Rather than being diagnostic criteria for determining simply whether a population is "regular," they should be treated as descriptive statistics that convey variation in the factors that influence neuronal positioning. We subsequently apply multiple spatial statistics to the analysis of eight different mosaics in the mouse retina, demonstrating conspicuous variability in the degree of patterning present, from essentially random to notably regular. This variability in patterning has both a developmental as well as a functional significance, reflecting the rules governing the positioning of different types of neurons as the architecture of the retina is assembled, and the distinct mechanisms by which they regulate dendritic growth to generate their characteristic coverage and connectivity.

We recently identified PKN1 as a developmentally active gatekeeper of the transcription factor neuronal differentiation-2 (NeuroD2) in several brain areas. Since NeuroD2 plays an important role in amacrine cell (AC) and retinal ganglion cell (RGC) type formation, we aimed to study the expression of NeuroD2 in the postnatal retina of WT and Pkn1−/− animals, with a particular focus on these two cell types. We show that PKN1 is broadly expressed in the retina and that the gross retinal structure is not different between both genotypes. Postnatal retinal NeuroD2 levels were elevated upon Pkn1 knockout, with Pkn1−/− retinae showing more NeuroD2+ cells in the lower portion of the inner nuclear layer. Accordingly, immunohistochemical analysis revealed an increased amount of AC in postnatal and adult Pkn1−/− retinae. There were no differences in horizontal cell, bipolar cell, glial cell and RGC numbers, nor defective axon guidance to the optic chiasm or tract upon Pkn1 knockout. Interestingly, we did, however, see a specific reduction in SMI-32+ α-RGC in Pkn1−/− retinae. These results suggest that PKN1 is important for retinal cell type formation and validate PKN1 for future studies focusing on AC and α-RGC specification and development.

Background: Loss of substantia nigra dopaminergic cells and alpha-synuclein (α-syn)-rich intraneuronal deposits within the central nervous system are key hallmarks of Parkinson’s disease (PD). Levodopa (L-DOPA) is the current gold-standard treatment for PD. This study aimed to evaluate in vivo retinal changes in a transgenic PD model of α-syn overexpression and the effect of acute levodopa (L-DOPA) treatment. Methods: Anaesthetised 6-month-old mice expressing human A53T alpha-synuclein (HOM) and wildtype (WT) control littermates were intraperitoneally given 20 mg/kg L-DOPA (50 mg levodopa, 2.5 mg benserazide) or vehicle saline (n = 11–18 per group). In vivo retinal function (dark-adapted full-field ERG) and structure (optical coherence tomography, OCT) were recorded before and after drug treatment for 30 min. Ex vivo immunohistochemistry (IHC) on flat-mounted retina was conducted to assess tyrosine hydroxylase (TH) positive cell counts (n = 7–8 per group). Results: We found that photoreceptor (a-wave) and bipolar cell (b-wave) ERG responses (p < 0.01) in A53T HOM mice treated with L-DOPA grew in amplitude more (47 ± 9%) than WT mice (16 ± 9%) treated with L-DOPA, which was similar to the vehicle group (A53T HOM 25 ± 9%; WT 19 ± 7%). While outer retinal thinning (outer nuclear layer, ONL, and outer plexiform layer, OPL) was confirmed in A53T HOM mice (p < 0.01), L-DOPA did not have an ameliorative effect on retinal layer thickness. These findings were observed in the absence of changes to the number of TH-positive amacrine cells across experiment groups. Acute L-DOPA treatment transiently improves visual dysfunction caused by abnormal alpha-synuclein accumulation. Conclusions: These findings deepen our understanding of dopamine and alpha-synuclein interactions in the retina and provide a high-throughput preclinical framework, primed for translation, through which novel therapeutic compounds can be objectively screened and assessed for fast-tracking PD drug discovery.

Blood vessels in the central nervous system (CNS) develop unique features, but the contribution of CNS neurons to regulating those features is not fully understood. We report that inhibiting spontaneous cholinergic activity or reducing starburst amacrine cell numbers prevents invasion of endothelial cells into the deep layers of the retina and causes blood-retinal-barrier (BRB) dysfunction in mice. Vascular endothelial growth factor (VEGF), which drives angiogenesis, and Norrin, a Wnt ligand that induces BRB properties, are decreased after activity blockade. Exogenous VEGF restores vessel growth but not BRB function, whereas stabilizing beta-catenin in endothelial cells rescues BRB dysfunction but not vessel formation. We further identify that inhibiting cholinergic activity reduces angiogenesis during oxygen-induced retinopathy. Our findings demonstrate that neural activity lies upstream of VEGF and Norrin, coordinating angiogenesis and BRB formation. Neural activity originating from specific neural circuits may be a general mechanism for driving regional angiogenesis and barrier formation across CNS development.

Directional responses in retinal ganglion cells are generated in large part by direction-selective release of γ-aminobutyric acid from starburst amacrine cells onto direction-selective ganglion cells (DSGCs). The excitatory inputs to DSGCs are also widely reported to be direction-selective, however, recent evidence suggests that glutamate release from bipolar cells is not directional, and directional excitation seen in patch-clamp analyses may be an artifact resulting from incomplete voltage control. Here, we test this voltage-clamp-artifact hypothesis in recordings from 62 ON-OFF DSGCs in the rabbit retina. The strength of the directional excitatory signal varies considerably across the sample of cells, but is not correlated with the strength of directional inhibition, as required for a voltage-clamp artifact. These results implicate additional mechanisms in generating directional excitatory inputs to DSGCs.

Many types of retinal neuron modulate the distribution of their processes to ensure a uniform coverage of the retinal surface. Dendritic field area, for instance, is inversely related to the variation in cellular density for many cell types, observed either across retinal eccentricity or between different strains of mice that differ in cell number. Dopaminergic amacrine (DA) cells, by contrast, have dendritic arbors that bear no spatial relationship to the presence of their immediate homotypic neighbors, yet it remains to be determined whether their coverage upon the retina, as a population, is conserved across variation in their total number. The present study assessed the overall density of the dopaminergic plexus in the inner plexiform layer in the presence of large variation in the total number of DA cells, as well as their retinal dopamine content, to determine whether either of these features is conserved. We first compared these traits between two strains of mice (C57BL/6J and A/J) that exhibit a two-fold difference in DA cell number. We subsequently examined these same traits in littermate mice for which the pro-apoptotic Bax gene was either intact or knocked out, yielding a five-fold difference in DA cell number. In both comparisons, we found greater plexus density and DA content in the strain or condition with the greater number of DA cells. The population of DA cells, therefore, does not appear to self-regulate its process coverage to achieve a constant density as the DA mosaic is established during development, nor its functional dopamine content in maturity.

Cell-intrinsic factors, in conjunction with environmental signals, guide migration, differentiation, and connectivity during early development of neuronal circuits. Within the retina, inhibitory starburst amacrine cells (SBACs) comprise ON types with somas in the ganglion cell layer (GCL) and dendrites stratifying narrowly in the inner half of the inner plexiform layer (IPL) and OFF types with somas in the inner nuclear layer (INL) and dendrites stratifying narrowly in the outer half of the IPL. The transcription factor Sox2 is crucial to this subtype specification. Without Sox2, many ON-type SBACs destined for the GCL settle in the INL while many that reach the GCL develop bistratified dendritic arbors. This study asked whether ON-type SBACs in Sox2-conditional knockout retinas exhibit selective connectivity only with ON-type bipolar cells or their bistratified morphology allows them to connect to both ON and OFF bipolar cells. Physiological data demonstrate that these cells receive ON and OFF excitatory inputs, indicating that the ectopically stratified dendrites make functional synapses with bipolar cells. The excitatory inputs were smaller and more transient in Sox2-conditional knockout compared with wild type; however, inhibitory inputs appeared largely unchanged. Thus dendritic stratification, rather than cellular identification, may be the major factor that determines ON vs. OFF connectivity. NEW & NOTEWORTHY Conditional knockout of the transcription factor Sox2 during early embryogenesis converts a monostratifying starburst amacrine cell into a bistratifying starburst cell. Here we show that these bistratifying starburst amacrine cells form functional synaptic connections with both ON and OFF bipolar cells. This suggests that normal ON vs. OFF starburst connectivity may not require distinct molecular specification. Proximity alone may be sufficient to allow formation of functional synapses.

Starburst amacrine cell (SAC) morphology is considered central to retinal direction selectivity. In Sema6A-/- mice, SAC dendritic arbors are smaller and no longer radially symmetric, leading to a reduction in SAC dendritic plexus density. Sema6A-/- mice also have a dramatic reduction in the directional tuning of retinal direction-selective ganglion cells (DSGCs). Here we show that the loss of DSGC tuning in Sema6A-/- mice is due to reduced null direction inhibition, even though strong asymmetric SAC-DSGC connectivity and SAC dendritic direction selectivity are maintained. Hence, the reduced coverage factor of SAC dendrites leads specifically to a loss of null direction inhibition. Moreover, SAC dendrites are no longer strictly tuned to centrifugal motion, indicating that SAC morphology is critical in coordinating synaptic connectivity and dendritic integration to generate direction selectivity.

The present study has taken advantage of publicly available cell type specific mRNA expression databases in order to identify potential genes participating in the development of retinal AII amacrine cells. We profile two such genes, Delta/Notch-like EGF repeat containing (Dner) and nuclear factor I/A (Nfia), that are each heavily expressed in AII amacrine cells in the mature mouse retina, and which conjointly identify this retinal cell population in its entirety when using antibodies to DNER and NFIA. DNER is present on the plasma membrane, while NFIA is confined to the nucleus, consistent with known functions of each of these two proteins. DNER also identifies some other subsets of retinal ganglion and amacrine cell types, along with horizontal cells, while NFIA identifies a subset of bipolar cells as well as Muller glia and astrocytes. During early postnatal development, NFIA labels astrocytes on the day of birth, AII amacrine cells at postnatal (P) day 5, and Muller glia by P10, when horizontal cells also transiently exhibit NFIA immunofluorescence. DNER, by contrast, is present in ganglion and amacrine cells on P1, also labeling the horizontal cells by P10. Developing AII amacrine cells exhibit accumulating DNER labeling at the dendritic stalk, labeling that becomes progressively conspicuous by P10, as it is in maturity. This developmental time course is consistent with a prospective role for each gene in the differentiation of AII amacrine cells.

The orderly spacing of retinal neurons is commonly regarded as a characteristic feature of retinal nerve cell populations. Exemplars of this property include the horizontal cells and the cholinergic amacrine cells, where individual cells minimize the proximity to like-type neighbors, yielding regularity in the patterning of their somata. Recently, two types of retinal bipolar cells in the mouse retina were shown to exhibit an order in their somal patterning no different from density-matched simulations constrained by soma size but being otherwise randomly distributed. The present study has now extended this finding to a type of retinal amacrine cell, the AII amacrine cell. Voronoi domain analysis revealed the patterning in the population of AII amacrine somata to be no different from density-matched and soma-size-constrained random simulations, while analysis of the density recovery profile showed AII amacrine cells to exhibit a minimal intercellular spacing identical to that for those random simulations: AII amacrine somata were positioned side-by-side as often as chance would predict. Regularity indexes and packing factors (PF) were far lower than those achieved by either the horizontal cells or cholinergic amacrine cells, with PFs also being comparable to those derived from the constrained random simulations. These results extend recent findings that call into question the widespread assumption that all types of retinal neurons are assembled as regular somal arrays, and have implications for the way in which AII amacrine cells must distribute their processes to ensure a uniform coverage of the retinal surface.

Rbfox1 is a splicing regulator that has been associated with various neurological conditions such as autism spectrum disorder, mental retardation, epilepsy, attention-deficit/hyperactivity disorder and schizophrenia. We show that in adult rodent retinas, Rbfox1 is expressed in all types of retinal ganglion cells (RGCs) and in certain subsets of amacrine cells (ACs), within the inner nuclear (INL) and ganglion cell (GCL) layers. In the INL, all Rbfox1-positive cells were colocalized with GABAergic ACs, however not all GABAergic ACs were immunostained for Rbfox1. In the GCL, a vast majority of GABAergic dACs were Rbfox1-immunopositive. Furthermore, all cholinergic starburst ACs (SACs) in the INL (type a) and in the GCL (type b) were Rbfox1 positive. The expression of Rbfox1 in the retina significantly overlapped with expression of Rbfox2, another member of Rbfox family of proteins. Rbfox2, in addition to RGCs and ACs, was also expressed in horizontal cells. In developing retinas at E12 and E15, Rbfox1 is localized to the cytoplasm of differentiating RGCs and ACs. Between P0 and P5, Rbfox1 subcellular localization switched from cytoplasmic to predominantly nuclear. Downregulation of Rbfox1 in adult Rbfox1loxP/loxP mice had no detectable effect on retinal gross morphology. However, the visual cliff test revealed marked abnormalities of depth perception of these animals. RNA sequencing of retinal transcriptomes of control and Rbfox1 knockout animals identified a number of Rbfox1-regulated genes that are involved in establishing neuronal circuits and synaptic transmission, including Vamp1, Vamp2, Snap25, Trak2, and Slc1A7, suggesting the role of Rbfox1 in facilitating synaptic communications between ACs and RGCs.

Rbfox1 is a multifunctional RNA binding protein that regulates various aspects of RNA metabolism important for neuronal differentiation and normal physiology. Rbfox1 has been associated with neurodevelopmental and neurological conditions as well as age-related neurodegenerative diseases such as Alzheimer's and Parkinson's. We have shown that in mammalian retinas Rbfox1 is expressed in retinal ganglion cells (RGCs) and in amacrine cells (ACs). This study investigates the effect of advanced age (22-month-old mice) on visual function, retinal morphology and survival of injured retinal ganglion cells (RGC) in Rbfox1 knockout (KO) animals. A visual cliff test, which was used to evaluate visual function, showed that 22-month old Rbfox1 KO mice have profound depth perception deficiency. Retinal gross morphology in these animals appeared to be normal. Optic nerve crush (ONC) induced axonal injury resulted in approximately 50% of RGC loss in both Rbfox1 KO and age-matched control animals: the average RGC densities in uninjured control and Rbfox1 KO animals were 6274 ± 1673 cells/mm2 and 6004 ± 1531 cells/mm2, respectively, whereas 1 week after ONC, RGC numbers in the retinas of control and Rbfox1 KO mice were reduced to 2998 ± 858 cells/mm2 and 3036 ± 857 cells/mm2, respectively (Rbfox1 KO vs. Rbfox1 KO + ONC, p < 0.0001 and control vs. control + ONC, p < 0.0001). No significant difference between RGC numbers in Rbfox1 KO + ONC and age-matched control + ONC animals was observed, suggesting that Rbfox1 has no effect on the survival of injured RGCs. Interestingly, however, contrary to a commonly accepted view that the number of RGCs in old (18 month of age) compared to young animals is reduced by approximately 40%, the RGC densities in 22-month-old mice in this study were similar to those of 4-month-old counterparts. [ABSTRACT FROM AUTHOR]

In the developing retina, as in other regions of the CNS, neural progenitors give rise to individual cell types during discrete temporal windows. Pax6 is expressed in retinal progenitor cells (RPCs) throughout the course of retinogenesis, and has been shown to be required during early retinogenesis for generation of most early-born cell types. In this study, we examined the function of Pax6 in postnatal mouse retinal development. We found that Pax6 is essential for the generation of late-born interneurons, while inhibiting photoreceptor differentiation. Generation of bipolar interneurons requires Pax6 expression in RPCs, while Pax6 is required for the generation of glycinergic, but not for GABAergic or non-GABAergic-non-glycinergic (nGnG) amacrine cell subtypes. In contrast, overexpression of either full-length Pax6 or its 5a isoform in RPCs induces formation of cells with nGnG amacrine features, and suppresses generation of other inner retinal cell types. Moreover, overexpression of both Pax6 variants prevents photoreceptor differentiation, most likely by inhibiting Crx expression. Taken together, these data show that Pax6 acts in RPCs to control differentiation of multiple late-born neuronal cell types.

RNA-binding proteins (RBPs) play central roles in post-transcriptional gene regulation. However, the function of RBP in retinal progenitor cell differentiation and synaptic signal transmission are largely unexplored. Previously we have shown that Elavl2 regulates amacrine cell (AC) differentiation during retinogenesis, by directly binding to Nr4a2 and Barhl2. Elavl2 is expressed in early neuronal progenitors to mature neurons, and Elavl4 expression begins slightly later, during cortical neuron development as a paralog. Here, Retinal-specific Elavl2 and Elavl4 double knockout mice were made to further explore the role of Elavl2 and Elavl4 in retinal development and signal transduction. We disclose that Elavl4 binds to Satb1 to regulate Neurod1, then promoting retinal progenitor and amacrine cells differentiation. We were also surprised to find that Elavl2 interacted with GABA B receptors at the RNA and protein levels. In conclusion, Elavl2 and Elavl4 regulate amacrine cells differentiation through different pathways, leading to decreased scotopic vision. Our findings reveal the roles of Elavl2 and Elavl4 in retinal amacrine cells differentiation in modulating visual functions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Retinal bipolar cells spread their dendritic arbors to tile the retinal surface, extending them to the tips of the dendritic fields of their homotypic neighbors, minimizing dendritic overlap. Such uniform nonredundant dendritic coverage of these populations would suggest a degree of spatial order in the properties of their somal distributions, yet few studies have examined the patterning in retinal bipolar cell mosaics. The present study examined the organization of two types of cone bipolar cells in the mouse retina, the Type 2 cells and the Type 4 cells, and compared their spatial statistical properties with those of the horizontal cells and the cholinergic amacrine cells, as well as to random simulations of cells matched in density and constrained by soma size. The Delauney tessellation of each field was computed, from which nearest neighbor distances and Voronoi domain areas were extracted, permitting a calculation of their respective regularity indexes (RIs). The spatial autocorrelation of the field was also computed, from which the effective radius and packing factor (PF) were determined. Both cone bipolar cell types were found to be less regular and less efficiently packed than either the horizontal cells or cholinergic amacrine cells. Furthermore, while the latter two cell types had RIs and PFs in excess of those for their matched random simulations, the two types of cone bipolar cells had spatial statistical properties comparable to random distributions. An analysis of single labeled cone bipolar cells revealed dendritic arbors frequently skewed to one side of the soma, as would be expected from a randomly distributed population of cells with dendrites that tile. Taken together, these results suggest that, unlike the horizontal cells or cholinergic amacrine cells which minimize proximity to one another, cone bipolar cell types are constrained only by their physical size.

Amacrine cells, inhibitory interneurons of the retina, feature synaptic inputs and outputs in close proximity throughout their dendritic trees, making them notable exceptions to prototypical somato-dendritic integration with output transmitted via axonal action potentials. The extent of dendritic compartmentalization in amacrine cells with widely differing dendritic tree morphology, however, is largely unexplored. Combining compartmental modeling, dendritic Ca2+ imaging, targeted microiontophoresis and multielectrode patch-clamp recording (voltage and current clamp, capacitance measurement of exocytosis), we investigated integration in the AII amacrine cell, a narrow-field electrically coupled interneuron that participates in multiple, distinct microcircuits. Physiological experiments were performed with in vitro slices prepared from retinas of both male and female rats. We found that the morphology of the AII enables simultaneous local and global integration of inputs targeted to different dendritic regions. Local integration occurs within spatially restricted dendritic subunits and narrow time windows and is largely unaffected by the strength of electrical coupling. In contrast, global integration across the dendritic tree occurs over longer time periods and is markedly influenced by the strength of electrical coupling. These integrative properties enable AII amacrines to combine local control of synaptic plasticity with location-independent global integration. Dynamic inhibitory control of dendritic subunits is likely to be of general importance for amacrine cells, including cells with small dendritic trees, as well as for inhibitory interneurons in other regions of the CNS. [ABSTRACT FROM AUTHOR]

In cold-blooded vertebrates such as zebrafish, Müller glial cells (MGs) readily proliferate to replenish lost retinal neurons. In mammals, however, MGs lack regenerative capability as they do not spontaneously re-enter the cell cycle unless the retina is injured. Here, we show that gene transfer of β-catenin in adult mouse retinas activates Wnt signaling and MG proliferation without retinal injury. Upstream of Wnt, deletion of GSK3β stabilizes β-catenin and activates MG proliferation. Downstream of Wnt, β-catenin binds to the Lin28 promoter and activates transcription. Deletion of Lin28 abolishes β-catenin-mediated effects on MG proliferation, and Lin28 gene transfer stimulates MG proliferation. We further demonstrate that let-7 miRNAs are critically involved in Wnt/Lin28-regulated MG proliferation. Intriguingly, a subset of cell-cycle-reactivated MGs express markers for amacrine cells. Together, these results reveal a key role of Wnt-Lin28-let7 miRNA signaling in regulating proliferation and neurogenic potential of MGs in the adult mammalian retina.

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 a non-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.

Understanding the function of modulatory interneuron networks is a major challenge, because such networks typically operate over long spatial scales and involve many neurons of different types. Here, we use an indirect electrical imaging method to reveal the function of a spatially extended, recurrent retinal circuit composed of two cell types. This recurrent circuit produces peripheral response suppression of early visual signals in the primate magnocellular visual pathway. We identify a type of polyaxonal amacrine cell physiologically via its distinctive electrical signature, revealed by electrical coupling with ON parasol retinal ganglion cells recorded using a large-scale multi-electrode array. Coupling causes the amacrine cells to fire spikes that propagate radially over long distances, producing GABA-ergic inhibition of other ON parasol cells recorded near the amacrine cell axonal projections. We propose and test a model for the function of this amacrine cell type, in which the extra-classical receptive field of ON parasol cells is formed by reciprocal inhibition from other ON parasol cells in the periphery, via the electrically coupled amacrine cell network.

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 a non-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.

The starburst amacrine cell in the mouse retina presents an opportunity to examine the precise role of sensory input location on neuronal computations. Using visual receptive field mapping, glutamate uncaging, two-photon Ca(2+) imaging, and genetic labeling of putative synapses, we identify a unique arrangement of excitatory inputs and neurotransmitter release sites on starburst amacrine cell dendrites: the excitatory input distribution is skewed away from the release sites. By comparing computational simulations with Ca(2+) transients recorded near release sites, we show that this anatomical arrangement of inputs and outputs supports a dendritic mechanism for computing motion direction. Direction-selective Ca(2+) transients persist in the presence of a GABA-A receptor antagonist, though the directional tuning is reduced. These results indicate a synergistic interaction between dendritic and circuit mechanisms for generating direction selectivity in the starburst amacrine cell.

An inner retinal microcircuit composed of dopamine (DA)-containing amacrine cells and melanopsin-containing, intrinsically photosensitive retinal ganglion cells (M1 ipRGCs) process information about the duration and intensity of light exposures, mediating light adaptation, circadian entrainment, pupillary reflexes, and other aspects of non-image-forming vision. The neural interaction is reciprocal: M1 ipRGCs excite DA amacrine cells, and these, in turn, feed inhibition back onto M1 ipRGCs. We found that the neuropeptide somatostatin [somatotropin release inhibiting factor (SRIF)] also inhibits the intrinsic light response of M1 ipRGCs and postulated that, to tune the bidirectional interaction of M1 ipRGCs and DA amacrine cells, SRIF amacrine cells would provide inhibitory modulation to both cell types. SRIF amacrine cells, DA amacrine cells, and M1 ipRGCs form numerous contacts. DA amacrine cells and M1 ipRGCs express the SRIF receptor subtypes sst(2A) and sst4 respectively. SRIF modulation of the microcircuit was investigated with targeted patch-clamp recordings of DA amacrine cells in TH-RFP mice and M1 ipRGCs in OPN4-EGFP mice. SRIF increases K(+) currents, decreases Ca(2+) currents, and inhibits spike activity in both cell types, actions reproduced by the selective sst(2A) agonist L-054,264 (N-[(1R)-2-[[[(1S*,3R*)-3-(aminomethyl)cyclohexyl]methyl]amino]-1-(1H-indol-3-ylmethyl)-2-oxoethyl]spiro[1H-indene-1,4'-piperidine]-1'-carboxamide) in DA amacrine cells and the selective sst4 agonist L-803,087 (N(2)-[4-(5,7-difluoro-2-phenyl-1H-indol-3-yl)-1-oxobutyl]-L-arginine methyl ester trifluoroacetate) in M1 ipRGCs. These parallel actions of SRIF may serve to counteract the disinhibition of M1 ipRGCs caused by SRIF inhibition of DA amacrine cells. This allows the actions of SRIF on DA amacrine cells to proceed with adjusting retinal DA levels without destabilizing light responses by M1 ipRGCs, which project to non-image-forming targets in the brain.

Signal processing within the retina is generally mediated by graded potentials, whereas output is conveyed by action potentials transmitted along optic nerve axons. Among retinal neurons, amacrine cells seem to be an exception to this general rule, as several types generate voltage‐gated Na+ (Nav) channel‐dependent action potentials. The AII, a narrow‐field, bistratified axon‐less amacrine cell found in mammalian retinas, displays a unique process that resembles an axon initial segment (AIS), with expression of Nav channels colocalized with the cytoskeletal protein ankyrin‐G, and generates action potentials. As the role of spiking in AIIs is uncertain, we hypothesized that the morphological properties of the AIS‐like process could provide information relevant for its functional importance, including potential pre‐ and/or postsynaptic connectivity. For morphological analysis, we injected AII amacrine cells in slices with fluorescent dye and immunolabeled the slices for ankyrin‐G. Subsequently, this enabled us to reliably identify AII‐type processes among ankyrin‐G‐labeled processes in wholemount retina. We systematically analyzed the laminar localization, spatial orientation, and distribution of the AIS‐like processes as a function of retinal eccentricity. In the horizontal plane, the processes displayed no preferred orientation and terminal endings were randomly distributed. In the vertical plane, the processes displayed a horizontal preference, but also ascended and descended into the inner nuclear layer and proximal inner plexiform layer, respectively. These results suggest that the AII amacrine AIS‐like process is unlikely to take part in conventional synaptic connections, but may instead be adapted to respond to volume neurotransmission by means of extrasynaptic receptors. [ABSTRACT FROM AUTHOR]

Transgenic mouse lines are essential tools for understanding the connectivity, physiology and function of neuronal circuits, including those in the retina. This report compares transgene expression in the retina of a tyrosine hydroxylase (TH)-red fluorescent protein (RFP) mouse line with three catecholamine-related Cre recombinase mouse lines [TH-bacterial artificial chromosome (BAC)-, TH-, and dopamine transporter (DAT)-Cre] that were crossed with a ROSA26-tdTomato reporter line. Retinas were evaluated and immunostained with commonly used antibodies including those directed to TH, GABA and glycine to characterize the RFP or tdTomato fluorescent-labeled amacrine cells, and an antibody directed to RNA-binding protein with multiple splicing to identify ganglion cells. In TH-RFP retinas, types 1 and 2 dopamine (DA) amacrine cells were identified by their characteristic cellular morphology and type 1 DA cells by their expression of TH immunoreactivity. In the TH-BAC-, TH-, and DAT-tdTomato retinas, less than 1%, ∼ 6%, and 0%, respectively, of the fluorescent cells were the expected type 1 DA amacrine cells. Instead, in the TH-BAC-tdTomato retinas, fluorescently labeled AII amacrine cells were predominant, with some medium diameter ganglion cells. In TH-tdTomato retinas, fluorescence was in multiple neurochemical amacrine cell types, including four types of polyaxonal amacrine cells. In DAT-tdTomato retinas, fluorescence was in GABA immunoreactive amacrine cells, including two types of bistratified and two types of monostratified amacrine cells. Although each of the Cre lines was generated with the intent to specifically label DA cells, our findings show a cellular diversity in Cre expression in the adult retina and indicate the importance of careful characterization of transgene labeling patterns. These mouse lines with their distinctive cellular labeling patterns will be useful tools for future studies of retinal function and visual processing.

Much of the computational power of the retina derives from the activity of amacrine cells, a large and diverse group of GABAergic and glycinergic inhibitory interneurons. Here, we identify an ON-type orientation-selective, wide-field, polyaxonal amacrine cell (PAC) in the rabbit retina and demonstrate how its orientation selectivity arises from the structure of the dendritic arbor and the pattern of excitatory and inhibitory inputs. Excitation from ON bipolar cells and inhibition arising from the OFF pathway converge to generate a quasi-linear integration of visual signals in the receptive field center. This serves to suppress responses to high spatial frequencies, thereby improving sensitivity to larger objects and enhancing orientation selectivity. Inhibition also regulates the magnitude and time course of excitatory inputs to this PAC through serial inhibitory connections onto the presynaptic terminals of ON bipolar cells. This presynaptic inhibition is driven by graded potentials within local microcircuits, similar in extent to the size of single bipolar cell receptive fields. Additional presynaptic inhibition is generated by spiking amacrine cells on a larger spatial scale covering several hundred microns. The orientation selectivity of this PAC may be a substrate for the inhibition that mediates orientation selectivity in some types of ganglion cells. Significance statement: The retina comprises numerous excitatory and inhibitory circuits that encode specific features in the visual scene, such as orientation, contrast, or motion. Here, we identify a wide-field inhibitory neuron that responds to visual stimuli of a particular orientation, a feature selectivity that is primarily due to the elongated shape of the dendritic arbor. Integration of convergent excitatory and inhibitory inputs from the ON and OFF visual pathways suppress responses to small objects and fine textures, thus enhancing selectivity for larger objects. Feedback inhibition regulates the strength and speed of excitation on both local and wide-field spatial scales. This study demonstrates how different synaptic inputs are regulated to tune a neuron to respond to specific features in the visual scene.

Ribbon synapses convey sustained and phasic excitatory drive within retinal microcircuits. However, the properties of retinal inhibitory synapses are less well known. AII-amacrine cells are interneurons in the retina that exhibit large glycinergic synapses at their dendritic lobular appendages. Using membrane capacitance measurements, we observe robust exocytosis elicited by the opening of L-type Ca(2+) channels located on the lobular appendages. Two pools of synaptic vesicles were detected: a small, rapidly releasable pool and a larger and more slowly releasable pool. Depending on the stimulus, either paired-pulse depression or facilitation could be elicited. During early postnatal maturation, the coupling of the exocytosis Ca(2+)-sensor to Ca(2+) channel becomes tighter. Light-evoked depolarizations of the AII-amacrine cell elicited exocytosis that was graded to light intensity. Our results suggest that AII-amacrine cell synapses are capable of providing both phasic and sustained inhibitory input to their postsynaptic partners without the benefit of synaptic ribbons.