Category Archives: Connectomics

Circuit Remodeling In Retinal Degeneration

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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.

Abstract:

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.

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.

2-nm Resolution Anatomy of Retinal Neuro-Glial-Vascular Architecture

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This abstract was presented today, May 2th at the 2016 Association for Research in Vision and Opthalmology (ARVO) meetings in Seattle, Washington by Jefferson R. Brown, Rebecca L. Pfeiffer, Crystal Sigulinsky, Felix Vazquez-Chona, Daniel Emrich, Bryan W. Jones, Robert E. Marc.

Abstract Number: 995

Author Block: Jefferson R. Brown1 , Rebecca L. Pfeiffer1 , Crystal Sigulinsky1 , Felix Vazquez-Chona1 , Daniel Emrich1 , Bryan W. Jones1 , Robert E. Marc1 
1 Dept of Ophthalmology, University of Utah, Salt Lake City, Utah, United States

Disclosure Block:Jefferson R. Brown, None; Rebecca L. Pfeiffer, None; Crystal Sigulinsky, None; Felix Vazquez-Chona, None; Daniel Emrich, None; Bryan W. Jones, None; Robert E. Marc, Signature Immunologics (Code I (Personal Financial Interest) )

Purpose:Retinal vasculature is strongly affected by degenerative pathologies and in turn, may also contribute to their progression. However, much of what we understand about the normal, healthy interaction between neurons, glia, and blood vessels at the ultrastructural level is limited to single section electron microscopy. The technology of serial section transmission electron microscopy (ssTEM) extends the high definition of TEM imaging into three dimensions to create volumes, allowing for more thorough visualization and analysis of the vascular-glial-neuronal complex.

Methods:RC2 is a 40TB ssTEM volume of over 1,400 horizontal sections of retinal tissue derived from an adult female C57BL/6J mouse. The tissue sample is 250 um in diameter and spans the outer nuclear layer to the vitreal surface. Baseline resolution is 2.18nm per pixel. Visualization, navigation and metadata annotations of the database are made via the Viking software suite.

Results:Much of the retinal vascular basement membrane directly contacts Muller cells. In the ganglion cell layer, direct basement membrane contact with astrocytes is frequent. Microglia commonly contact the basement membrane, and occasionally direct contact of neurons onto basement membrane was observed. Full 3D reconstruction of all vascular pathways with associated endothelia and pericytes within the volume was completed, demonstrating that all the retinal capillary layers are continuous with one another [Figure].

Conclusions:The presence of occasional direct neuronal contact onto vascular basement membrane supports earlier work by Ochs and colleagues (2000) and suggests the blood-retina barrier does not universally involve retinal glia. However, since such contacts are extremely sparse, it remains to be seen whether this finding has biologic significance, though their existence suggests significance. The RC2 volume is a valuable resource to aid in discovery of defining characteristics of wild type neurovascular architecture.


The intro figure is a side view of reconstruction of all vasculature within the RC2 volume. Vessels at the top of the figure correspond to the outer plexiform layer, while those at the bottom correspond to the ganglion cell layer. This capillary plexus is one continuous structure. Visualization by VikingView software.

Store-Operated Calcium Entry In Müller Glia Is Controlled By Synergistic Activation Of TRPC And Orai Channels

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We have a new publication out as collaborators with colleages, Store-Operated Calcium Entry In Müller Glia Is Controlled By Synergistic Activation Of TRPC And Orai Channels authored by Tünde Molnár, Oleg Yarishkin, Peter Barabas, Anthony Iuso, Bryan W. Jones, Robert Marc, Tam Phuong, and David Krizaj.

Bonus, we got the cover!  The image was created by Tam Phuong.

 

Significance: Store-operated Ca2+ signaling represents a major signaling pathway and source of cytosolic Ca2+ in astrocytes. Here, we show that the store-operated response in Müller cells, radial glia that perform key structural, signaling, osmoregulatory and mechanosensory functions within the retina, is mediated through synergistic activation of TRPC and Orai channels. The endfoot disproportionately expresses the depletion sensor STIM1, contains an extraordinarily high density of ER cisternae that shadow neuronal, astrocyte, vascular and axonal structures, interface with mitochondria but also originates SOCE-induced transcellular Ca2+ waves that propagate glial excitation into the proximal retina. These results identify a molecular mechanism that underlies complex interactions between the plasma membrane and calcium stores and contributes to radial glial function, regulation and response to mechanical stress.

Abstract: The endoplasmic reticulum (ER) is at the epicenter of astrocyte Ca2+ signaling. We sought to identify the molecular mechanism underlying store-operated calcium entry (SOCE) that repletes ER Ca2+ stores in mouse Müller cells. Store depletion, induced through blockade of sequestration transporters in Ca2+-free saline, induced synergistic activation of canonical transient receptor potential (TRPC1) and Orai channels. Store-operated TRPC1 channels were identified by their electrophysiological properties, pharmacological blockers and ablation of the Trpc1 gene. ICRAC (Ca2+ release-activated) currents were identified by ion permeability, voltage-dependence and sensitivity to selective Orai antagonists Synta66 and GSK7975A. Depletion-evoked calcium influx was initiated at the Müller endfoot and apical process, triggering centrifugal propagation of Ca2+ waves into the cell body. EM analysis of the endfoot compartment showed high-density ER cisternae that shadow retinal ganglion cell (RGC) somata and axons, protoplasmic astrocytes, vascular endothelial cells and ER-mitochondrial contacts at the vitreal surface of the endfoot. The mouse retina expresses transcripts encoding both Stim and all known Orai genes; Müller glia predominantly express STIM1 whereas STIM2 is mainly confined to the outer plexiform and retinal ganglion cell layers. Elimination of TRPC1 facilitated Müller gliosis induced by the elevation of intraocular pressure (IOP), suggesting that TRPC channels might play a neuroprotective role during mechanical stress. These findings expand the current knowledge about store-operated signaling in astroglia, as well as calcium signaling pathways in Müller astroglia and functional roles these cells play in retinal physiology and pathology.

Seasonal And Post-Trauma Remodeling Of The Ground Squirrel Retina

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We have a new publication out, Seasonal and post-trauma remodeling in cone-dominant ground squirrel retina authored by Dana Merriman, Ben Sajdak, Wei Li and Bryan W. Jones.

Abstract:

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.

The AII Amacrine Cell Connectome: A Dense Network Hub

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AII-connectome

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.

 

A Synaptic Basis for Small World Network Design in the ON Inner Plexiform Layer of the Rabbit Retina

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Bipolar cells_

This abstract was presented today at the 2014 Association for Research in Vision and Opthalmology (ARVO) meetings in Orlando, Florida by J Scott Lauritzen, Noah T. Nelson, Crystal L. Sigulinsky, Nathan Sherbotie, John Hoang, Rebecca L. Pfeiffer, James R. Anderson, Carl B. Watt, Bryan W. Jones and Robert E. Marc.

Purpose: Converging evidence suggests that large- and intermediate-scale neural networks throughout the nervous system exhibit small world’ design characterized by high local clustering of connections yet short path length between neuronal modules (Watts & Strogatz 1998 Nature; Sporns et al.2004 Trends in Cog Sci). It is suspected that this organizing principle scales to local networks (Ganmor et al. 2011 J Neurosci; Sporns 2006 BioSystems) but direct observation of synapses and local network topologies mediating small world design has not been achieved in any neuronal tissue. We sought direct evidence for synaptic and topological substrates that instantiate small world network architectures in the ON inner plexiform layer (IPL) of the rabbit retina. To test this we mined ≈ 200 ON cone bipolar cells (BCs) and ≈ 500 inhibitory amacrine cell (AC) processes in the ultrastructural rabbit retinal connectome (RC1).

Methods: BC networks in RC1 were annotated with the Viking viewer and explored via graph visualization of connectivity and 3D rendering (Anderson et al. 2011 J Microscopy). Small molecule signals embedded in RC1 e.g. GABA glycine and L-glutamate combined with morphological reconstruction and connectivity analysis allow for robust cell classification. MacNeil et al. (2004 J Comp Neurol) BC classification scheme used for clarity.

Results: Homocellular BC coupling (CBb3::CBb3 CBb4::CBb4 CBb5::CBb5) and within-class BC inhibitory networks (CBb3 → AC –| CBb3 CBb4 → AC –| CBb4 CBb5 → AC –| CBb5) in each ON IPL strata form laminar-specific functional sheets with high clustering coefficients. Heterocellular BC coupling (CBb3::CBb4 CBb4::CBb5 CBb3::CBb5) and cross-class BC inhibitory networks (CBb3 → AC –| CBb4 CBb4 → AC –| CBb3 CBb4 → AC –| CBb5 CBb5 → AC –| CBb4 CBb3 → AC –| CBb5 CBb5 → AC –| CBb3) establish short synaptic path lengths across all ON IPL laminae.

Conclusions: The retina contains a greater than expected number of synaptic hubs that multiplex parallel channels presynaptic to ganglion cells. The results validate a synaptic basis (ie. direct synaptic connectivity) and local network topology for the small world architecture indicated at larger scales providing neuroanatomical plausibility of this organization for local networks and are consistent with small world design as a fundamental organizing principle of neural networks on multiple spatial scales.

Support:  NIH EY02576 (RM), NIH EY015128 (RM), NSF 0941717 (RM), NIH EY014800 Vision Core (RM), RPB CDA (BWJ), Thome AMD Grant (BWJ).

Synapse Classification And Localization In Electron Micrographs

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Synapse-classification_

We have a new publication, Synapse Classification And Localization In Electron Micrographs in Pattern Recognition Letters.  Authors are: Vignesh Jagadeesh, James Anderson, Bryan W. Jones, Robert Marc, Steven Fisher and B.S. Manjunath.

Abstract:  Classification and detection of biological structures in Electron Micrographs (EM) is a relatively new large scale image analysis problem. The primary challenges are in modeling diverse visual characteristics and development of scalable techniques. In this paper we propose novel methods for synapse detection and localization, an important problem in connectomics. We first propose an attribute based descriptor for characterizing synaptic junctions. These descriptors are task specific, low dimensional and can be scaled across large image sizes. Subsequently, techniques for fast localization of these junctions are proposed. Experimental results on images acquired from a mammalian retinal tissue compare favorably with state of the art descriptors used for object detection.