Tag Archives: bipolar cell

Preprint: Neural Circuit Revision in Retinal Remodeling, A Pathoconnectomics Approach

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We have a new preprint out, Neural Circuit Revision in Retinal Remodeling, A Pathoconnectomics Approach.

Authors: Rebecca L Pfeiffer, Jeebika Dahal, Crystal L Sigulinsky, James R Anderson, Isabel A Barrera, Jia-Hui Yang, Olivia Haddadin, Alexis R Houser, Jessica C Garcia, Bryan William Jones

Abstract: The Aii glycinergic amacrine cell (Aii) plays a central role in bridging rod pathways with cone pathways, enabling an increased dynamic range of vision from scotopic to photopic ranges. The Aii integrates scotopic signals via chemical synapses from rod bipolar cells (RodBCs) onto the arboreal processes of Aii ACs, injecting signals into ON-cone bipolar cells (CBbs) via gap junctions with Aiis on the arboreal processes and the waist of the Aii ACs. The CBbs then carry this information to ON and OFF ganglion cell classes. In addition, the Aii is involved in the surround inhibition of OFF cone bipolar cells (CBas) through glycinergic chemical synapses from Aii ACs onto CBas. We have previously shown changes in RodBC connectivity as a consequence of rod photoreceptor degeneration in a pathoconnectome of early retinal degeneration: RPC1. Here, we evaluated the impact of rod photoreceptor degeneration on the connectivity of the Aii to determine the impacts of photoreceptor degeneration on the downstream network of the neural retina and its suitability for integrating therapeutic interventions as rod photoreceptors are lost. Previously, we reported that in early retinal degeneration, prior to photoreceptor cell loss, Rod BCs make pathological gap junctions with Aiis. Here, we further characterize this altered connectivity and additional shifts in both the excitatory drive and gap junctional coupling of Aiis in retinal degeneration, along with discussion of the broader impact of altered connectivity networks. New findings reported here demonstrate that Aiis make additional gap junctions with CBas increasing the number of BC classes that make pathological gap junctional connectivity with Aiis in degenerating retina. In this study, we also report that the Aii, a tertiary retinal neuron alters their synaptic contacts early in photoreceptor degeneration, indicating that rewiring occurs in more distant members of the retinal network earlier in degeneration than was previously predicted. This rewiring impacts retinal processing, presumably acuity, and ultimately its ability to support therapeutics designed to restore image-forming vision. Finally, these Aii alterations may be the cellular network level finding that explains one of the first clinical complaints from human patients with retinal degenerative disease, an inability to adapt back and forth from photopic to scotopic conditions.

Distinctive Synaptic Structural Motifs Link Excitatory Retinal Interneurons To Diverse Postsynaptic Partner Types

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We have a new manuscript from the lab in Cell Reports, Distinctive Synaptic Structural Motifs Link Excitatory Retinal Interneurons To Diverse Postsynaptic Partner Types. This manuscript is in collaboration with the first author, Wan-Qing Yu @wanqing_yu, then co-authors Rachael Swanstrom, Crystal L. Sigulinsky @CSigulinsky, Richard M. Ahlquist, along with Sharm Knecht, myself Bryan W. Jones @BWJones. David M. Berson, and Rachel O. Wong. The PDF is here.

Abstract:
Neurons make converging and diverging synaptic connections with distinct partner types. Whether synapses involving separate partners demonstrate similar or distinct structural motifs is not yet well understood. We thus used serial electron microscopy in mouse retina to map output synapses of cone bipolar cells (CBCs) and compare their structural arrangements across bipolar types and postsynaptic partners. Three presynaptic configurations emerge—single-ribbon, ribbonless, and multiribbon synapses. Each CBC type exploits these arrangements in a unique combination, a feature also found among rabbit ON CBCs. Though most synapses are dyads, monads and triads are also seen. Altogether, mouse CBCs exhibit at least six motifs, and each CBC type uses these in a stereotypic pattern. Moreover, synapses between CBCs and particular partner types appear biased toward certain motifs. Our observations reveal synaptic strategies that diversify the output within and across CBC types, potentially shaping the distinct functions of retinal microcircuits.

Network Architecture of Gap Junctional Coupling among Parallel Processing Channels in the Mammalian Retina

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We have a new manuscript out in The Journal of Neuroscience, Network Architecture of Gap Junctional Coupling among Parallel Processing Channels in the Mammalian Retina.

Authors: Crystal L. Sigulinsky @CLSigulinsky, James R. Anderson, Ethan Kerzner @EthanKerzner, Christopher N. Rapp @ChrisNRapp, Rebecca L. Pfeiffer @BeccaPfeiffer19, Taryn M. Rodman, Daniel P. Emrich, Kevin D. Rapp, Noah T. Nelson @nooneelseinhere, J. Scott Lauritzen, Miriah Meyer@miriah_meyer, Robert E. Marc @robertmarc60, and Bryan W. Jones @BWJones.

Abstract: Gap junctions are ubiquitous throughout the nervous system, mediating critical signal transmission and integration, as well as emergent network properties. In mammalian retina, gap junctions within the Aii amacrine cell-ON cone bipolar cell (CBC) network are essential for night vision, modulation of day vision, and contribute to visual impairment in retinal degenerations, yet neither the extended network topology nor its conservation is well established. Here, we map the network contribution of gap junctions using a high-resolution connectomics dataset of an adult female rabbit retina. Gap junctions are prominent synaptic components of ON CBC classes, constituting 5%–25% of all axonal synaptic contacts. Many of these mediate canonical transfer of rod signals from Aii cells to ON CBCs for night vision, and we find that the uneven distribution of Aii signals to ON CBCs is conserved in rabbit, including one class entirely lacking direct Aii coupling. However, the majority of gap junctions formed by ON CBCs unexpectedly occur between ON CBCs, rather than with Aii cells. Such coupling is extensive, creating an interconnected network with numerous lateral paths both within, and particularly across, these parallel processing streams. Coupling patterns are precise with ON CBCs accepting and rejecting unique combinations of partnerships according to robust rulesets. Coupling specificity extends to both size and spatial topologies, thereby rivaling the synaptic specificity of chemical synapses. These ON CBC coupling motifs dramatically extend the coupled Aii-ON CBC network, with implications for signal flow in both scotopic and photopic retinal networks during visual processing and disease.

OFF-layer Branches of ON Cone Bipolar Cells in Early Retinal Degeneration

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This abstract was presented today, April 8th at the 2019 Association for Research in Vision and Opthalmology (ARVO) meetings in Vancouver, Canada by Jessica C. Garcia, Rebecca L. Pfeiffer, Crystal L. Sigulinsky, James R. Anderson, Daniel P. Emrich, Jeebika Dahal, Hope Morrison, Kevin D. Rapp, Jia-Hui Yang, Carl B. Watt, Mineo Kondo, Hiroko Terasaki, Robert E. Marc and Bryan W. Jones.

Full resolution version here.

Purpose: Cone bipolar cells are customarily classified into superclasses of ON (ON-BCs) and OFF (OFF-BCs). ON or OFF specialization is further segregated by stratification within the inner plexiform layer. Retinal degeneration induces negative plasticity termed remodeling, that includes aberrant neurite extension from multiple cell types (rewiring) and ON-BCs switching their glutamate receptors to match that of OFF-BCs (reprogramming). Previous analysis in healthy retina shows that ON-BCs can make small ribbon synapses in the descending axon or, less frequently, simple single branch projections in the OFF layer. What impact remodeling has on these OFF-layer branches is unknown. In this study, we compare OFF branches from ON-BCs in a connectome of early retinal degeneration (RPC1) to our healthy retinal connectome (RC1).

Methods: Retinal tissues selected for RC1 and RPC1 were collected post-mortem from a 13 month old Dutch-belted healthy female rabbit and a male 10 month old transgenic P347L rabbit model of autosomal dominant retinitis pigmentosa, respectively. RPC1 shows signs of remodeling including rod outer-segment degeneration and aberrant neurite extension. Tissues were fixed in mixed aldehydes and subsequently osmicated, dehydrated, resin embedded, and sectioned at 90 nm (RC1) or 70 nm (RPC1). Sections were placed on formvar grids, stained, and imaged at 2nm/px on a JEOL JEM-1400 TEM using SerialEM software. 1 section was reserved from every 30 for Computational Molecular Phenotyping, and probed for small molecules. Both volumes were evaluated using the Viking software suite.

Results: Ribbons of ON-BCs formed in the OFF layer branches have been previously described to contact glycinergic amacrine cells (ACs), GABAergic ACs, ON ganglion cells, and intrinsically photosensitive ganglion cells. Initial analysis of OFF branches of ON-BCs in RPC1 demonstrate more complex branching than in RC1 and increased number of synapses on these branches. In contrast to the inconsistent OFF layer branch stratification observed in RC1, the OFF branches in RPC1 appear to stratify at a similar level. Evaluation of synaptic partners is ongoing.

Conclusions: Increased complexity and number of synapses found in the OFF branches of some ON-BCs ultimately may represent ON network corruption. Exploring synaptic partners will reveal potential network alterations in retinal degenerative disease.

Rod Bipolar Cell Networks In A Retinal Pathoconnectome

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We presented a poster on Rod Bipolar Cell Networks In A Retinal Pathoconnectome at the 2019 HHMI Connectomics meeting in Berlin today. Downsampled PDF of poster here.

Authors: Rebecca L. Pfeiffer, James R. Anderson, Daniel P. Emrich, Jeebika Dahal, Crystal L. Sigulinsky, Jia-Hui Yang, Kevin D. Rapp, Carl B. Watt, Jessica C. Garcia, Mineo Kondo, Hiroko Terasaki, Robert E. Marc, and Bryan W. Jones.

Abstract: Ultrastructural connectomics has allowed for precise identification of neural network topologies in retina, exposing synaptic connectivity associated with specific pathways involved in neural retinal processing. In pathological degenerate retina such as retinitis pigmentosa (RP), retinal remodeling emerges as a phenomenon through a series of negative plasticity events originating from neural deafferentation initiated by photoreceptor degeneration. Early stages of remodeling include glial changes, GluR receptor alterations (reprogramming), and rewiring of retinal networks. The connectivities initiated by these processes are currently unknown. To address this problem, we have created an ultrastructural pathoconnnectome of early retinal remodeling in a rabbit model of retinitis pigmentosa, Retinal Pathoconnectome 1 (RPC1).

Mapping the network architecture of gap junctional coupling among parallel processing channels in the mammalian retina

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We presented a poster on Mapping the network architecture of gap junctional coupling among parallel processing channels in the mammalian retina at the 2019 HHMI Connectomics meeting in Berlintoday. Downsampled PDF of poster here.

Authors: Crystal L. Sigulinsky, James R. Anderson, Ethan Kerzner, Christopher N. Rapp, Rebecca L. Pfeiffer, Daniel P. Emrich, Kevin D. Rapp, Noah T. Nelson, J. Scott Lauritzen, Miriah Meyer, Robert E. Marc, and Bryan W. Jones.

Abstract: Electrical synapses are fundamental components of neural networks. Gap junctions provide the anatomical basis for electrical synapses and are prevalent throughout the neural retina with essential roles in signal transmission. Gap junctions within and between the parallel processing channels afforded by retinal bipolar cells have been reported or predicted, but their roles, partners, and patterns remain largely unknown. Here, we took advantage of the high resolution of Retinal Connectome 1 (RC1) to reconstruct ON cone bipolar cells (CBCs) and map their coupling topologies.

Heterocellular Coupling Between Amacrine Cells and Ganglion Cells

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We have a new paper out In Frontiers in Neural Circuits, Heterocellular Coupling Between Amacrine Cells and Ganglion Cells. This manuscript preprint was published in BioRxiv.

Authors: Robert E. Marc, Crystal Lynn Sigulinsky, Rebecca L. Pfeiffer, Daniel Emrich, James Russel Anderson and Bryan William Jones.

Abstract: All superclasses of retinal neurons, including bipolar cells (BCs), amacrine cells (ACs) and ganglion cells (GCs), display gap junctional coupling. However, coupling varies extensively by class. Heterocellular AC coupling is common in many mammalian GC classes. Yet, the topology and functions of coupling networks remains largely undefined. GCs are the least frequent superclass in the inner plexiform layer and the gap junctions mediating GC-to-AC coupling (GC::AC) are sparsely arrayed amidst large cohorts of homocellular AC::AC, BC::BC, GC::GC and heterocellular AC::BC gap junctions. Here, we report quantitative coupling for identified GCs in retinal connectome 1 (RC1), a high resolution (2 nm) transmission electron microscopy-based volume of rabbit retina. These reveal that most GC gap junctions in RC1 are suboptical. GC classes lack direct cross-class homocellular coupling with other GCs, despite opportunities via direct membrane contact, while OFF alpha GCs and transient ON directionally selective (DS) GCs are strongly coupled to distinct AC cohorts. Integrated small molecule immunocytochemistry identifies these as GABAergic ACs (γ+ ACs). Multi-hop synaptic queries of RC1 connectome further profile these coupled γ+ ACs. Notably, OFF alpha GCs couple to OFF γ+ ACs and transient ON DS GCs couple to ON γ+ ACs, including a large interstitial amacrine cell, revealing matched ON/OFF photic drive polarities within coupled networks. Furthermore, BC input to these γ+ ACs is tightly matched to the GCs with which they couple. Evaluation of the coupled versus inhibitory targets of the γ+ ACs reveals that in both ON and OFF coupled GC networks these ACs are presynaptic to GC classes that are different than the classes with which they couple. These heterocellular coupling patterns provide a potential mechanism for an excited GC to indirectly inhibit nearby GCs of different classes. Similarly, coupled γ+ ACs engaged in feedback networks can leverage the additional gain of BC synapses in shaping the signaling of downstream targets based on their own selective coupling with GCs. A consequence of coupling is intercellular fluxes of small molecules. GC::AC coupling involves primarily γ+ cells, likely resulting in GABA diffusion into GCs. Surveying GABA signatures in the GC layer across diverse species suggests the majority of vertebrate retinas engage in GC::γ+ AC coupling.

Coupling Architecture Of The Aii/ON Cone Bipolar Cell Network In The Degenerate Retina

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Crystal Sigulinsky, a post-doc in the lab, presented her work on “coupling architecture of the
Aii/ON cone bipolar cell network in the degenerate retina” at the RD2018 meeting in Killarney, Ireland today.  Authors are: Crystal L Sigulinsky, Rebecca L Pfeiffer, James R Anderson, Jeebika Dahal, Hope Morrison, Daniel P. Emrich, Jessica C Garcia, Jia-Hui Yang, Carl B. Watt, Kevin D. Rapp, Mineo Kondo, Hiroko Terasaki, Robert E. Marc, and Bryan W. Jones.

Purpose: Retinal network hyperactivity within degenerative retinal networks is a component of the disease process with implications for therapeutic interventions for blinding diseases that depend upon the surviving retinal network. Connexin36-containing gap junctions centered on the Aii amacrine cell network appear to mediate the aberrant signaling observed in mouse models of retinal degeneration. However, it remains unclear whether this hyperactivity reflects changes in the underlying circuitry or dysfunction/dysregulation of the normative circuitry. Mapping retinal circuitry in the ultrastructural rabbit Retinal Connectome, RC1, has revealed Aii network topologies explicitly involving gap junctions. In addition to canonical Aii-to-Aii and Aii-to-ON cone bipolar cell (CBC) coupling, we describe pervasive in- and cross-class coupling motifs among ON CBCs that extend and dramatically expand the coupled Aii network topologies. Since virtually every gap junction in the inner plexiform layer contains Connexin36, these circuits likely participate in the aberrant signaling of degenerate retinas. This study examines these Aii and ON CBC coupling motifs in Retinal PathoConnectome 1 (RPC1), an ultrastructural pathoconnectome of a rabbit model of retinitis pigmentosa.

Approach: RPC1 is a 2nm/pixel resolution volume of retina from a 10 month old, transgenic P347L rabbit model of autosomal dominant retinitis pigmentosa in early phase 1 retinal remodeling, a time point where cone and rod photoreceptors are still present, albeit going through cell stress. RPC1 spans the vitreous to basal outer nuclear layer and was built by automated transmission electron microscopy and computational assembly. ON CBCs, Aii amacrine cells, and their coupling partners were annotated using the Viking application and explored with 3D rendering and graph visualization of connectivity. Gap junctions were validated by 0.25 nm resolution recapture with goniometric tilt when necessary. Motifs were compared to those discovered in RC1. RC1 is a 2 nm resolution, 0.25 mm diameter volume of a light-adapted adult female Dutch Belted rabbit retina spanning the ganglion cell through inner nuclear layers.

Conclusions: RPC1 shows degeneration of rod outer segments, Müller cell hypertrophy and neuronal sprouting, characteristic of early stage retinal degeneration and phase 1 remodeling, when retinal hyperactivity and its reliance on gap junctional coupling has likely already initiated and human patients would still have some vision. All major coupling motifs (Aii-to-Aii, Aii-to-ON CBC, and ON CBC-to-ON CBC) were observed. Preliminary examinations indicate that several ON CBC classes retained their class-specific coupling profiles, accepting and rejecting specific combinations of Aii and ON CBC class partnerships. However, recent findings reveal aberrant partnerships in the coupled network, including both loss of prominent motifs and acquisition of novel ones. Thus, clear aberrant morphological and synaptic changes have been identified in RPC1, including changes in the coupling specificity and gap junction distributions of both Aii amacrine cells and ON CBCs (Figure 6). This suggests that the Aii/ON CBC circuit topology is already altered during early phase 1 remodeling, with substantial implications for therapeutic interventions in human subjects. The full coupling network is actively being examined and progress has begun on RPC2, a second pathoconnectome for examining later, phase 2 remodeling in this same model.

An almost full size poster available here in pdf format.

The Rod-Cone Crossover Connectome of Mammalian Bipolar Cells

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We have a new publication out (direct link), The rod-cone crossover connectome of mammalian bipolar cells authored by Scott Lauritzen, Crystal Sigulinsky, James Anderson, Michael Kalloniatis, Noah Nelson, Danny Emrich, Chris Rapp, Nicolas McCarthy, Ethan Kerzner, Mariah Meyer, Bryan W. Jones, and Robert Marc.

Abstract: The basis of cross-suppression between rod and cone channels has long been an enigma. Using rabbit retinal connectome RC1, we show that all cone bipolar cell (BC) classes inhibit rod BCs via amacrine cell (AC) motifs (C1-6); that all cone BC classes are themselves inhibited by AC motifs (R1-5, R25) driven by rod BCs. A sparse symmetric AC motif (CR) is presynaptic and postsynaptic to both rod and cone BCs. ON cone BCs of all classes drive inhibition of rod BCs via motif C1 wide-field GABAergic ACs (γACs) and motif C2 narrow field glycinergic ON ACs (GACs). Each rod BC receives ≈ 10 crossover AC synapses and each ON cone BC can target ≈ 10 or more rod BCs via separate AC processes. OFF cone BCs mediate monosynaptic inhibition of rod BCs via motif C3 driven by OFF γACs and GACs and disynaptic inhibition via motifs C4 and C5 driven by OFF wide-field γACs and narrow-field GACs, respectively. Motifs C4 and C5 form halos of 60-100 inhibitory synapses on proximal dendrites of AI γACs. Rod BCs inhibit surrounding arrays of cone BCs through AII GAC networks that access ON and OFF cone BC patches via motifs R1, R2, R4 R5 and a unique ON AC motif R3 that collects rod BC inputs and targets ON cone BCs. Crossover synapses for motifs C1, C4, C5 and R3 are 3-4x larger than typical feedback synapses, which may be a signature for synaptic winner-take-all switches.

Retinal Remodeling in Human Retinitis Pigmentosa

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We have a new publication out (Direct Link, Free Open Access), Retinal Remodeling in Human Retinitis Pigmentosa authored by Bryan W. Jones, Rebecca Pfeiffer, Drew Ferrell, Carl Watt, Michael Marmor and Robert Marc.

Abstract: Retinitis Pigmentosa (RP) in the human is a progressive, currently irreversible neural degenerative disease usually caused by gene defects that disrupt the function or architecture of the photoreceptors. While RP can initially be a disease of photoreceptors, there is increasing evidence that the inner retina becomes progressively disorganized as the outer retina degenerates. These alterations have been extensively described in animal models, but remodeling in humans has not been as well characterized. This study, using computational molecular phenotyping (CMP) seeks to advance our understanding of the retinal remodeling process in humans. We describe cone mediated preservation of overall topology, retinal reprogramming in the earliest stages of the disease in retinal bipolar cells, and alterations in both small molecule and protein signatures of neurons and glia. Furthermore, while Müller glia appear to be some of the last cells left in the degenerate retina, they are also one of the first cell classes in the neural retina to respond to stress which may reveal mechanisms related to remodeling and cell death in other retinal cell classes. Also fundamentally important is the finding that retinal network topologies are altered. Our results suggest interventions that presume substantial preservation of the neural retina will likely fail in late stages of the disease. Even early intervention offers no guarantee that the interventions will be immune to progressive remodeling. Fundamental work in the biology and mechanisms of disease progression are needed to support vision rescue strategies.