Category Archives: Ultrastructure

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.

Impact of Retinal Degeneration on Response of ON and OFF Cone Bipolar Cells to Electrical Stimulation

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We have a new manuscript from the lab in IEEE, Impact of Retinal Degeneration on Response of ON and OFF Cone Bipolar Cells to Electrical Stimulation. This manuscript is in collaboration with the Lazzi lab out of USC.  The first author, Shayan Farzad, Pragya Kosta, Ege Iseri, Steven T Walston, Jean-Marie C. Bouteiller,  Rebecca L. Pfeiffer @BeccaPfeiffer19, Crystal L. Sigulinsky @CSigulinsky, Jia-Hui Yang, Jessica C. Garcia, James R. Anderson, Bryan W. Jones @BWJones, and Gianluca Lazzi. The PDF is here.

Abstract: In retinal degenerative diseases, such as retinitis pigmentosa (RP) and age-related macular degeneration (AMD), the photoreceptors become stressed and start to degenerate in the early stages of the disease. Retinal prosthetic devices have been developed to restore vision in patients by applying electrical stimulation to the surviving retinal cells. However, these devices provide limited visual perception as the therapeutic interventions are generally considered in the later stages of the disease when only inner retinal layer cells are left. A potential treatment option for retinal degenerative diseases in the early stages can be stimulating bipolar cells, which receive presynaptic signals from photoreceptors. In this work, we constructed computational models of healthy and degenerated (both ON and OFF-type) cone bipolar cells (CBCs) with realistic morphologies extracted from connectomes of the healthy and early-stage degenerated rabbit retina. We examined these cells’ membrane potential and axon terminal calcium current differences when subjected to electrical stimulation. In addition, we investigated how differently healthy and degenerated cells behave with respect to various stimulation parameters, including pulse duration and cells’ distance from the stimulating electrode. The results suggested that regardless of the position of the OFF CBCs in the retina model, there is not a significant difference between the membrane potential of healthy and degenerate cells when electrically stimulated. However, the healthy ON CBC axon terminal membrane potential rising time-constant is shorter (0.29 ± 0.03 ms) than the degenerated cells (0.8 ± 0.07 ms). Moreover, the ionic calcium channels at the axon terminals of the cells have a higher concentration and higher current in degenerated cells (32.24 ± 6.12 pA) than the healthy cells (13.64 ± 2.88 pA) independently of the cell’s position.

Mitochondrial Transfer Between Inner Retinal Neurons

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This abstract was presented today, April 26th at the 2023 Association for Research in Vision and Opthalmology (ARVO) meetings in New Orleans, Louisiana by Selena Wirthlin, Crystal Sigulinsky, James Anderson, and Bryan William Jones.

Full resolution version here.

Purpose
Intercellular mitochondrial transfer has been reported across a variety of cells and tissues under both physiological and pathological conditions. Such transfer has shown broad therapeutic potential. The effectiveness of this therapy, however, is limited by a lack of understanding of the cellular and molecular mechanisms. Here, the ultrastructural features of mitochondrial transfer between inner retinal neurons discovered through retinal connectomics analysis is shown.

Methods
Retinal Connectome 2 (RC2) was built by automated transmission electron microscopy at ultrastructural (2nm/pixel) resolution. RC2 is a 0.25mm diameter volume of retina obtained from a 5-month-old female C57BL/6J mouse. The Viking application was used to visualize and annotate inter- and intracellular features of interest in the connectome.

Results
Exploration of RC2 revealed material transfer between apposing neural processes within the OFF subliminal of the inner plexiform layer. The transferred material can be defined as a mitochondria, confirmed by the presence of crustae. At the transfer site, a short, electron-dense 140-nm diameter tube with a curved cap tightly associated with the inner mitochondrial membrane of one neuritis extends into a vacuole within the apposing neuritis formed by the plasma membranes of the two cells. Thin cytoskeletal components consistent with actin microfilaments extend into the mitochondrion. Morphology and synaptology of the acceptor cell confirm it is an Aii amacrine cell, while preliminary findings suggest the donor cell is a type of ON/OFF ganglion cell.

Conclusions
These findings demonstrate active mitochondrial transfer between different classes of endogenous inner retinal neurons and suggests it may represent an important component of tissue homeostasis in the retina. Features of this transfer differ from previously reported mitochondrial transfer between photoreceptors upon transplantation, which may indicate cell type- or context-dependent differences in the cellular or molecular mechanisms. Our findings demonstrate active mitochondrial transfer between different classes of endogenous inner retinal neurons and suggest it may represent an important component of tissue homeostasis in the retina. Features of this transfer differ from previous reports by the Wallace and Pearson groups of material transfer between photoreceptors upon transplantation through tunneling nanotubes (Ortin- Martinez et al., 2021; Kalargyrou et al., 2021), which may indicate cell type- or context-dependent differences in the cellular or molecular mechanisms. Understanding these mechanisms could serve as a catalyst for development of novel therapeutics for disease in the retina and beyond.

Müller Cell Connectomics In Health And Disease

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This talk was presented today, April 25th at the 2023 Association for Research in Vision and Opthalmology (ARVO) meetings in New Orleans, Louisiana by Rebecca Pfeiffer as part of an ARVO Minisymposium Bryan William Jones organized.

Abstract: Muller cells are a critical component of retinal function and rapidly change metabolically and morphologically in retinal disease. Of Muller cell functions, many require close physical relationships between the Muller cell and the synapses of the neurons they support. Despite this required neuro-glial relationship, little is known about the direct contacts between Muller cells and synapses in healthy or diseased retinas. In order to address this, I use a connectomics/pathoconnectomics approach to reconstruct Muller cells and their neighboring synapses. The retinas evaluated are from a healthy rabbit, retinal connectome 1 (RC1), and from the P347L rabbit model of retinitis pigmentosa, retinal pathoconnectome 1 (RPC1). Preliminary data demonstrate an increase in endfoot entanglement in RPC1 when compared with RC1, and direct synaptic contact analysis of both connectomes is ongoing.

Species-Specific Connectivity In The Aii Connectome

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This talk was presented today, April 25th at the 2023 Association for Research in Vision and Opthalmology (ARVO) meetings in New Orleans, Louisiana by Crystal Sigulinsky as part of an ARVO Minisymposium organized by Bryan William Jones.

Abstract: Biomedical research relies heavily on animal models to study human disease and develop therapeutics. Understanding the architectural diversity in neural networks between humans and these model species is essential for choosing a relevant study model and interpreting conflicting results. Using comparative connectomics, we sought to map and compare the local neural network architecture of rabbit and mouse retinal Aii amacrine cells. This specialized narrow-field, multistratified, glycinergic interneuron has critical feedforward and feedback roles in both the photopic and scotopic retinal networks spanning the ON and OFF pathways, making it an ideal candidate for investigating species-specific differences in retinal networks. High-resolution, serial-section transmission electron microscopy (TEM) volumes of rabbit (RC1: female, 13- month, Dutch Belted) and mouse (RC2: female, 5-month, C57BL/6J) retinal tissue provided spatially-registered synaptic maps of Aii connectivity at directly comparable resolution and completeness. These reveal that despite species-specific morphologies, gross synaptology and compartmentalization appear conserved. Yet, rabbit and mouse Aii cells diverge in the weighting of their partnerships, most notably in their coupling profiles. Opposing biases in gap junction partnerships and their respective sizing rules indicate a greater relative output by mouse Aii cells to ON pathways than in rabbit. However, a unique topological conformation for a subset of conventional presynapses formed by Aii cell lobular dendrites with species-specific features and prevalence may influence signal output to specific partner classes within the OFF pathway and either nullify or exacerbate this difference in ON/OFF output. Additionally, rabbit Aii cells in RC1 showed greater Aii-Aii coupling than in mouse, which may suggest greater signal-to-noise compensation. Lastly, preliminary data suggest mouse Aii cells receive greater excitatory, but not inhibitory input/feedback from the OFF pathway than in rabbit. Together these data indicate that precise neural circuit architectures diverge between species and require detailed, comprehensive mapping to begin to dissect potential influence on signal flow.

Structural Motifs Of Excitatory Synapses In The Mammalian Retina

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This abstract was presented today, April 24th at the 2023 Association for Research in Vision and Opthalmology (ARVO) meetings in New Orleans, Louisiana by Taylor Otterness, Crystal Sigulinsky, James Anderson, and Bryan William Jones.

Full resolution version here.

Purpose
Connectivity within the nervous system is precise and disruptions lead to degraded performance and disease, yet the rules that govern connectivity remain unknown. Recent efforts reveal that different types of cone bipolar cells in the neural retina show preferences in the selection and frequency of presynaptic structure types used for signal transmission. However, it is not yet known how these differences are related to the quantity or type of postsynaptic partner. We used Retinal Connectome 1 (RC1) to analyze the synaptic output of rabbit CBb6 cells, a type of ON cone bipolar cell that forms excitatory synapses via diverse presynaptic structure types, to identify patterns in how these cells interact with their postsynaptic partners.

Methods
RC1 is a 0.25 mm diameter volume sampled from mid-peripheral retina of a 13 month old female Dutch-Belted rabbit, serially sectioned at 70 nm, and imaged at ultrastructural resolution (2nm/px) using transmission electron microscopy. Postsynaptic partners of CBb6 cell 6156’s presynaptic structures were annotated using the Viking Viewer for Connectomics. Statistical analyses were conducted in Microsoft Excel and investigated further with 3D rendering and graph visualization of connectivity.

Results
The factors tracked for comparison included presynaptic structure type, target number, and postsynaptic partner type. Multiribbon synapses of CBb6 cell 6156 trended towards having a greater number of output partners, with a greater proportion of dyads than monads. Despite this, triads and quadrads were only found opposing single ribbon synapses. As the different presynaptic structure types may differ in the strength of neurotransmitter release (ribbonless < single ribbon < multiribbon), these findings are inconsistent with scaling of output to the number of postsynaptic targets. Both amacrine cells (AC) and ganglion cells (GC) are postsynaptic partners of 6156. However, single ribbon and ribbonless structures appear biased towards AC only targets, while multiribbon synapses appear biased toward mixed AC and GC targets.

Conclusions
Target type relationships appear more important than the number of targets in determining presynaptic structure type in CBb6. Future efforts will examine size differences of postsynaptic structures and presynaptic ribbon size, and even compare across bipolar cell classes, in order to provide further insight on the connectivity rules underlying excitatory synapses.

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.

Current Perspective on Retinal Remodeling: Implications for Therapeutics

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We have a new paper out of the lab, a perspectives paper on Retinal Remodeling: Implications for Therapeutics. (pdf here).

Authors are Rebecca L. Pfeiffer @BeccaPfeiffer19, and Bryan W. Jones @BWJones.

Abstract: The retinal degenerative diseases retinitis pigmentosa and age-related macular degeneration are a leading cause of irreversible vision loss. Both present with progressive photoreceptor degeneration that is further complicated by processes of retinal remodeling. In this perspective, we discuss the current state of the field of retinal remodeling and its implications for vision-restoring therapeutics currently in development. Here, we discuss the challenges and pitfalls retinal remodeling poses for each therapeutic strategy under the premise that understanding the features of retinal remodeling in totality will provide a basic framework with which therapeutics can interface. Additionally, we discuss the potential for approaching therapeutics using a combined strategy of using diffusible molecules in tandem with other vision-restoring therapeutics. We end by discussing the potential of the retina and retinal remodeling as a model system for more broadly understanding the progression of neurodegeneration across the central nervous system.

Peptide-Based Immunotherapy Against Oxidized Elastin Ameliorates Pathology In Mouse Model Of Smoke-Induced Ocular Injury

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We have a new collaborative manuscript out in iOVS, Peptide-Based Immunotherapy Against Oxidized Elastin Ameliorates Pathology In Mouse Model Of Smoke-Induced Ocular Injury. (pdf here)

Authors: Bärbel Rohrer, Nathaniel Parsons , Balasubramaniam Annamalai , Crystal Nicholson , Elisabeth Obert , Bryan Jones @BWJones, and Andrew D Dick.

Abstract:

Purpose: Age-related macular degeneration (AMD), the leading cause of blindness in western populations, is associated with an overactive complement system, and an increase in circulating antibodies against certain epitopes, including elastin. As loss of the elastin layer of Bruch’s membrane (BrM) has been reported in aging and AMD, we previously showed that immunization with elastin peptide oxidatively modified by cigarette smoke (ox-elastin), exacerbated ocular pathology in the smoke-induced ocular pathology (SIOP) model. Here we asked whether ox-elastin peptide-based immunotherapy (PIT) ameliorates damage.

Methods: C57BL/6J mice were injected with ox-elastin peptide at two doses via weekly subcutaneous administration, while exposed to cigarette smoke for 6 months. FcγR-/- and uninjected C57BL/6J mice served as controls. Retinal morphology was assessed by by electron microscopy, and complement activation, antibody deposition and mechanisms of immunological tolerance were assessed by Western blotting and ELISA.

Results: Elimination of Fcγ receptors, preventing antigen/antibody-dependent cytotoxicity, protected against SIOP. Mice receiving PIT with low dose ox-elastin (LD-PIT) exhibited reduced humoral immunity, reduced complement activation and IgG/IgM deposition in the RPE/choroid, and largely a preserved BrM. While there is no direct evidence of ox-elastin pathogenicity, LD-PIT reduced IFNγ and increased IL-4 within RPE/choroid. High dose PIT was not protective.

Conclusions: These data further support ox-elastin role in ocular damage in in part via elastin-specific antibodies, and support the corollary that PIT with ox-elastin attenuates ocular pathology. Overall, damage is associated with complement activation, antibody-dependent cell-mediated cytotoxicity, and altered cytokine signature.

Keywords: Age-related macular degeneration; Complement; Elastin; Peptide-based immunotherapy; Smoking.

Primary Cilia in Amacrine Cells in Retinal Development

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We have a new collaborative manuscript out in iOVS, Primary Cilia in Amacrine Cells in Retinal Development. (pdf here)

Authors: Ke Ning; Brent E. Sendayen; Tia J. Kowal; Biao Wang; Bryan W. Jones @BWJones; Yang Hu; and Yang Sun.

Abstract:

Purpose: Primary cilia are conserved organelles found in polarized cells within the eye that regulate cell growth, migration, and differentiation. Although the role of cilia in photoreceptors is well-studied, the formation of cilia in other retinal cell types has received little attention. In this study, we examined the ciliary profile focused on the inner nuclear layer of retinas in mice and rhesus macaque primates.

Methods: Retinal sections or flatmounts from Arl13b-Cetn2 tg transgenic mice were immunostained for cell markers (Pax6, Sox9, Chx10, Calbindin, Calretinin, ChaT, GAD67, Prox1, TH, and vGluT3) and analyzed by confocal microscopy. Primate retinal sections were immunostained for ciliary and cell markers (Pax6 and Arl13b). Optical coherence tomography (OCT) and ERGs were used to assess visual function of Vift88 mice.

Results: During different stages of mouse postnatal eye development, we found that cilia are present in Pax6-positive amacrine cells, which were also observed in primate retinas. The cilia of subtypes of amacrine cells in mice were shown by immunostaining and electron microscopy. We also removed primary cilia from vGluT3 amacrine cells in mouse and found no significant vision defects. In addition, cilia were present in the outer limiting membrane, suggesting that a population of Müller glial cells forms cilia.

Conclusions: We report that several subpopulations of amacrine cells in inner nuclear layers of the retina form cilia during early retinal development in mice and primates.