Our paper Rod-cone crossover connectome of mammalian bipolar cells has been republished in a special issue of The Journal Of Comparative Neurology, Retinal Special Issue I: Mammals.
Our paper Rod-cone crossover connectome of mammalian bipolar cells has been republished in a special issue of The Journal Of Comparative Neurology, Retinal Special Issue I: Mammals.
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.
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.
Rebecca Pfeiffer, a post-doc in the laboratory presented her work on “Rod Bipolar Cell Networks in Early Retinal Remodeling” as a platform presentation at the ISER 2018 meeting in Belfast, Northern Ireland.
Authors: Rebecca Pfeiffer, James R. Anderson, Daniel P. Emrich, Jeebika Dahal, Crystal L Sigulinsky, Hope AB Morrison, Jia-Hui Yang, Carl B. Watt, Kevin D. Rapp, Jessica C Garcia, Mineo Kondo, Hiroko Terasaki, Robert E. Marc, and Bryan W. Jones.
Abstract: Retinal remodeling is a form of negative plasticity that occurs as a consequence of retinal degenerative diseases. Part of retinal remodeling involves anomalous sprouting of processes, termed neurites. The synaptic structures and partners of the neurites are not yet defined, leading to uncertainty about the consistency of network motifs between healthy and degenerate retina. Our goal is to map out the identities and network relationships of bipolar cell networks using a connectomics strategy. Retinal connectomes or ultrastructural maps of neuronal connectivity have substantially contributed to our understanding of retinal network topology, providing ground truth against which pathological network topologies can be evaluated. We have generated the first pathoconnectome (RPC1), or connectome of pathological tissues, of early retinal remodeling at 2nm/pixel, and are currently investigating the impact of remodeling on network architecture.
The tissue for RPC1 was obtained from a 10mo transgenic P347L rabbit model of autosomal dominant retinitis pigmentosa. Tissue was fixed in mixed aldehydes, osmicated, dehydrated, embedded in epon resin, and sectioned at 70nm. Serial sections were placed on grids, stained, and imaged using a JEOL JEM-1400 TEM using SerialEM software. Every 30th section was reserved for computational molecular phenotyping (CMP), and probed for small molecules: glutamate, glutamine, glycine, GABA, taurine, glutathione; or TEM compatible proteins GFAP and GS. The pathoconnectome volume is explored and annotated using the Viking software suite.
RPC1 was selected as an example of early retinal remodeling, demonstrating Muller cell hypertrophy, metabolic dysregulation, and degeneration of rod outer segments, indicating phase 1 remodeling and neuronal sprouting. We have observed the presence of both cone pedicles and rod spherules within the OPL to be synaptically active with neurites from some rod bipolar cells forming functional synapses with both rod spherules and cone pedicles. These rod bipolar cells also exhibit structurally altered ribbon synapses. We are currently evaluating network motifs and comparing them to networks established from our previous connectome, RC1, generated from a healthy rabbit.
These findings allow us to evaluate and analyze the impact of retinal remodeling on retinal networks which may have important implications for therapeutic interventions being developed which rely on inner retina network integrity.
Rebecca Pfeiffer, a post-doc in the laboratory presented her work on “Pathoconnectome Analysis of Müller Cells in Early Retinal Remodeling” as a platform presentation at the RD2018 meeting in Killarney, Ireland.
Authors: Rebecca Pfeiffer, James R. Anderson, Daniel P. Emrich, Jeebika Dahal, Crystal L Sigulinsky, Hope AB Morrison, Jia-Hui Yang, Carl B. Watt, Kevin D. Rapp, Mineo Kondo, Hiroko Terasaki, Jessica C Garcia, Robert E. Marc, and Bryan W. Jones.
Purpose: Glia play important roles in neural system function. These roles include, but are not limited to: amino acid recycling, ion homeostasis, glucose transport, and removal of waste. During retinal degeneration, Muller cells, the primary macroglia of the retina, are one of the first cells to show metabolic and morphological alterations in response to retinal stress. The metabolic alterations observed in Muller cells appear to manifest in regions of photoreceptor degeneration; however, the precise mechanisms that govern these alterations in response to neuronal stress, synapse maintenance, or glia-glia interactions is currently unknown. This project aims to reconstruct Muller cells from a pathoconnectome of early retinal remodeling at 2nm/pixel with ultrastructural metabolic data to determine the relationship of structural and metabolic phenotypes between neighboring neurons and glia.
Methods: Retinal pathoconnectome 1 (RPC1) is the first connectome to be assembled from pathologic neural tissue (a pathoconnectome). The tissue selected for RPC1 was collected post mortem from a 10 month transgenic P347L rabbit model of autosomal dominant retinitis pigmentosa, fixed in 1% formaldehyde, 2.5% glutaraldehyde, 3% sucrose, and 1mM MgSO4 in cacodylate buffer (pH 7.4). The tissue was subsequently osmicated, dehydrated, resin embedded, and sectioned at 70nm. Sections were placed on formvar grids, stained, and imaged at 2nm/pixel on a JEOL JEM-1400 TEM using SerialEM software. 1 section was reserved from every 30 sections for CMP, where it was placed on a slide and probed for small molecules: glutamate, glutamine, glycine, GABA, taurine, glutathione; or TEM compatible proteins GFAP and GS. The pathoconnectome volume was evaluated and annotated using the Viking software suite.
Results: RPC1 demonstrates hallmarks of early retinal degeneration and remodeling, including the glial phenotypes of hypertrophy and metabolic variation between neighboring Muller cells. Early evaluation of these glia demonstrates variations in osmication in Muller cells as well as apparent encroachment of glial end-feet on one another. We are currently in the process of reconstructing multiple Muller cells within RPC1 and their neighboring neurons. Once complete, we will assess the relationship between Muller cell phenotype and the phenotypes of contacted neuronal and glial neighbors.
Conclusions: How neural-glial relationships are affected by retinal remodeling may help us understand why remodeling and neurodegeneration follow photoreceptor degeneration. In addition, determining these relationships during remodeling will be crucial to developing therapeutics with long-term success. RPC1 provides a framework to analyze these relationships in early retinal remodeling through ultrastructural reconstructions of all neurons and glia in an intact retina. These reconstructions, informed by quantitative metabolite labeling, will allow us to evaluate these neural-glial interactions more comprehensively than other techniques have previously allowed.
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.
This abstract was presented today, Monday, April 30th at the 2018 Association for Research in Vision and Opthalmology (ARVO) meetings in Honolulu, Hawaii by Rebecca Pfeiffer, Robert E. Marc, James R. Anderson, Daniel P. Emrich, Carl B. Watt, Jia-Hui Yang, Kevin D. Rapp, Jeebika Dahal, Mineo Kondo, Hiroko Terasaki, and Bryan W. Jones.
Retinal remodeling is a consequence of retinal degenerative disease, during which neurons sprout new neurites whose synaptic structures and partners are not yet defined. Simultaneously during remodeling, Müller cells (MCs) undergo structural and metabolic changes, whose impact on surrounding neurons is an active area of research. Retinal connectomes have elucidated and validated fundamental networks. These data provide further classification of neuronal types and subtypes and a precise framework for modeling of retinal function, based on ground truth networks. The creation of the first pathoconnectome (RPC1), a connectome from pathological retinal tissue, provides the opportunity to determine connectivites between neurons, while simultaneously evaluating glial remodeling. Computational Molecular Phenotyping (CMP) embedded within the ultrastructure provides metabolic factors of pathologies.
RPC1 was collected post-mortem from a 10mo TgP347L rabbit model of adRP, fixed in 1% FA, 2.5% GA, 3% sucrose, and 1mM MgSO4 in cacodylate buffer (pH 7.4). The tissue was osmicated, dehydrated, resin embedded, and sectioned at 70nm. Sections were placed on formvar grids, stained, and imaged on a JEOL JEM-1400 TEM using SerialEM. 1 section was reserved from every 30 section for CMP, where it was probed for small molecules: glutamate, glutamine, glycine, GABA, taurine, glutathione; or proteins GFAP and GS. RPC1 was evaluated using the Viking software suite.
RPC1 was chosen based on early features of retinal degeneration/remodeling: degeneration of rod OS, MC hypertrophy, and neuronal sprouting. RPC1 consists of 948 serial sections spanning the ONL to the vitreous, with a diameter of 90µm. We find dendrites extending from rod bipolar cells to cone pedicles, originally described in light microscopy, and active synaptic contacts. We also see alterations of synaptic structure in the IPL, and MC morphological changes affecting surface to volume and neuron/glial relationships. Network motifs are being actively investigated.
We observe many features of remodeling previously described using light microscopy, and confirm active synaptic contact. We also find synaptic structural features, not previously described. In addition, early evaluation of MC morphology demonstrates marked changes in MC shape and associations with nearby neurons and glia, which, combined with CMP, will be instrumental in understanding how MCs affect retinal remodeling.
This abstract was presented yesterday, April 29th at the 2018 Association for Research in Vision and Opthalmology (ARVO) meetings in Honolulu, Hawaii by Bryan W. Jones.
The retina is a complex, heterocellular tissue with most/all retinal cell classes becoming impacted or altered in retinitis pigmentosa (RP) and age-related macular degeneration (AMD) in a process called retinal remodeling. Defining disease and the stage-specific cytoarchitectural and metabolic responses in RP and AMD is critical for highlighting targets for intervention. We now know that negative plasticity and neural retinal remodeling occurs regardless of retinal insult in models of retinal degeneration as well as in human RP and in human AMD, revealing that no retinal disease fails to trigger remodeling and reprogramming.
Evidence in the literature over the past decade has improved our understanding into mechanisms of initial retinal degeneration and informed our understanding of the subsequent remodeling events in the neural retina that occur post-photoreceptor degeneration. Remodeling associated with retinal degeneration is intimately linked with insults that cause photoreceptor stress and eventually photoreceptor cell death. These phenomena result in reprogramming of cell types in retina followed by progressive neural degeneration akin to CNS neural degenerations involving both neuronal and glial classes. No cell class in the retina is spared from the effects of remodeling. The earliest cell classes involved in remodeling are horizontal, bipolar and Müller cells and the Müller glia are the last cell class left in the remodeling retina.
Our efforts are now focused on elucidating the precise wiring changes in retina, through the creation of pathological connectomes, or “patho-connectomes” to study precisely what the circuit topologies are, compared to normal topologies derived from Retinal Connectome 1 (RC1). Also, because temporal windows are critical to understanding when interventions may be possible, we are exploring when circuit topology revisions occur to understand their impact on information flow in the retina and their impact on rescues of vision loss. Precise circuit topologies in early retinal degenerative events is our first area of exploration with ultrastructural reconstructions of outer retinal neurons, bipolar cells and horizontal cells. Müller glia are also of intense interest as we are tracking the earliest metabolic and morphological changes in glia in response to retinal degenerations.
With a photoreceptor mosaic containing ∼85% cones, the ground squirrel is one of the richest known mammalian sources of these important retinal cells. It also has a visual ecology much like the human’s. While the ground squirrel retina is understandably prominent in the cone biochemistry, physiology, and circuitry literature, far less is known about the remodeling potential of its retinal pigment epithelium, neurons, macroglia, or microglia. This review aims to summarize the data from ground squirrel retina to this point in time, and to relate them to data from other brain areas where appropriate. We begin with a survey of the ground squirrel visual system, making comparisons with traditional rodent models and with human. Because this animal’s status as a hibernator often goes unnoticed in the vision literature, we then present a brief primer on hibernation biology. Next we review what is known about ground squirrel retinal remodeling concurrent with deep torpor and with rapid recovery upon re-warming. Notable here is rapidly-reversible, temperature-dependent structural plasticity of cone ribbon synapses, as well as pre- and post-synaptic plasticity throughout diverse brain regions. It is not yet clear if retinal cell types other than cones engage in torpor-associated synaptic remodeling. We end with the small but intriguing literature on the ground squirrel retina’s remodeling responses to insult by retinal detachment. Notable for widespread loss of (cone) photoreceptors, there is surprisingly little remodeling of the RPE or Müller cells. Microglial activation appears minimal, and remodeling of surviving second- and third-order neurons seems absent, but both require further study. In contrast, traumatic brain injury in the ground squirrel elicits typical macroglial and microglial responses. Overall, the data to date strongly suggest a heretofore unrecognized, natural checkpoint between retinal deafferentiation and RPE and Müller cell remodeling events. As we continue to discover them, the unique ways by which ground squirrel retina responds to hibernation or injury may be adaptable to therapeutic use.
We have a new publication in Frontiers in Neuroscience, The AII Amacrine Cell Connectome: A Dense Network Hub. Authors are Robert E. Marc, James R. Anderson, Bryan W. Jones, Crystal Sigulinsky and J. Scott Lauritzen.
Abstract: The mammalian AII retinal amacrine cell is a narrow-field, multistratified glycinergic neuron best known for its role in collecting scotopic signals from rod bipolar cells and distributing them to ON and OFF cone pathways in a crossover network via a combination of inhibitory synapses and heterocellular AII::ON cone bipolar cell gap junctions. Long considered a simple cell, a full connectomics analysis shows that AII cells possess the most complex interaction repertoire of any known vertebrate neuron, contacting at least 28 different cell classes, including every class of retinal bipolar cell. Beyond its basic role in distributing rod signals to cone pathways, the AII cell may also mediate narrow-field feedback and feedforward inhibition for the photopic OFF channel, photopic ON-OFF inhibitory crossover signaling, and serves as a nexus for a collection of inhibitory networks arising from cone pathways that likely negotiate fast switching between cone and rod vision. Further analysis of the complete synaptic counts for five AII cells shows that (1) synaptic sampling is normalized for anatomic target encounter rates; (2) qualitative targeting is specific and apparently errorless; and (3) that AII cells strongly differentiate partner cohorts by synaptic and/or coupling weights. The AII network is a dense hub connecting all primary retinal excitatory channels via precisely weighted drive and specific polarities. Homologs of AII amacrine cells have yet to be identified in non-mammalians, but we propose that such homologs should be narrow-field glycinergic amacrine cells driving photopic ON-OFF crossover via heterocellular coupling with ON cone bipolar cells and glycinergic synapses on OFF cone bipolar cells. The specific evolutionary event creating the mammalian AII scotopic-photopic hub would then simply be the emergence of large numbers of pure rod bipolar cells.
We have a new publication, Retinal Prosthetics, Optogenetics and Photoswitches in ACS Chemical Neuroscience. Authors are: Robert E. Marc, Rebecca L. Pfeiffer, and Bryan W. Jones.
Three technologies have emerged as therapies to restore light sensing to profoundly blind patients suffering from late-stage retinal degenerations: (1) retinal prosthetics, (2) optogenetics, and (3) chemical photoswitches. Prosthetics are the most mature and the only approach in clinical practice. Prosthetic implants require complex surgical intervention and provide only limited visual resolution but can potentially restore navigational ability to many blind patients. Optogenetics uses viral delivery of type 1 opsin genes from prokaryotes or eukaryote algae to restore light responses in survivor neurons. Targeting and expression remain major problems, but are potentially soluble. Importantly, optogenetics could provide the ultimate in high-resolution vision due to the long persistence of gene expression achieved in animal models. Nevertheless, optogenetics remains challenging to implement in human eyes with large volumes, complex disease progression, and physical barriers to viral penetration. Now, a new generation of photochromic ligands or chemical photoswitches (azobenzene-quaternary ammonium derivatives) can be injected into a degenerated mouse eye and, in minutes to hours, activate light responses in neurons. These photoswitches offer the potential for rapidly and reversibly screening the vision restoration expected in an individual patient. Chemical photoswitch variants that persist in the cell membrane could make them a simple therapy of choice, with resolution and sensitivity equivalent to optogenetics approaches. A major complexity in treating retinal degenerations is retinal remodeling: pathologic network rewiring, molecular reprogramming, and cell death that compromise signaling in the surviving retina. Remodeling forces a choice between upstream and downstream targeting, each engaging different benefits and defects. Prosthetics and optogenetics can be implemented in either mode, but the use of chemical photoswitches is currently limited to downstream implementations. Even so, given the high density of human foveal ganglion cells, the ultimate chemical photoswitch treatment could deliver cost-effective, high-resolution vision for the blind.