Category Archives: Connectomics

Chapter: Retinal Connectomics

We have a new chapter out in the Elsevier book series The Senses, 2021.

Authors are myself, Bryan W. Jones @BWJones and Robert E. Marc @robertmarc60.

Abstract: The retina is both a light sensor and a highly complex image-processing device – like supercomputers at the backs of eyes. The retina is also wonderfully compact with all circuitry (glia, neurons, synapses and gap junctions) required to compute sensory input, making it a convenient model for understanding the rest of the nervous system. This is also true for disease, with early evidence indicating retina may be a good model for studying progressive neural degenerative diseases. Modern ultrastructural approaches to the study of neural connections is a relatively new !eld has been termed “connectomics”. Connectomics approaches applied to the retina is termed retinal connectomics. These approaches are relatively new !elds that leverage modern technologies in light and ultrastructural imaging, computational storage, and data management to allow tracking of neuronal identity and connectivity, delivering a robust edge/node network map of circuit topologies. Understanding circuit topologies is critical to understanding how retinas process information, and how information processing is corrupted in disease. This chapter summarizes early history, discusses technical aspects of imaging connectomes, justi!es the importance of why connectomics approaches are important, particularly in retina, discusses what has been learned from early efforts in connectomics, and points the way to the next steps.

Please email me: bryan.jones@m.cc.utah.edu if you would like a pdf of the chapter.

 

A pathoconnectome of early neurodegeneration: Network changes in retinal degeneration

We have a new manuscript out in Experimental Eye Research, A pathoconnectome of early neurodegeneration: Network changes in retinal degeneration. (pdf here)

Authors: Rebecca L. Pfeiffer @BeccaPfeiffer19, James R. Anderson, Jeebika Dahal, Jessica C. Garcia, Jia-Hui Yang, Crystal L. Sigulinsky @CLSigulinsky, Kevin Rapp, Daniel P. Emrich, Carl B. Watt, Hope AB Johnstun, Alexis R. Houser, Robert E. Marc @robertmarc60, and Bryan W. Jones @BWJones.

Abstract: Connectomics has demonstrated that synaptic networks and their topologies are precise and directly correlate with physiology and behavior. The next extension of connectomics is pathoconnectomics: to map neural network synaptology and circuit topologies corrupted by neurological disease in order to identify robust targets for therapeutics. In this report, we characterize a pathoconnectome of early retinal degeneration. This pathoconnectome was generated using serial section transmission electron microscopy to achieve an ultrastructural connectome with 2.18nm/px resolution for accurate identification of all chemical and gap junctional synapses. We observe aberrant connectivity in the rod-network pathway and novel synaptic connections deriving from neurite sprouting. These observations reveal principles of neuron responses to the loss of network components and can be extended to other neurodegenerative diseases.

 

NeuroNex Grant

I am pleased to report that the The Marclab for Connectomics has been funded by the National Science Foundation with a 5 year grant as part of a large, international consortium to study synaptic weighting.  We are collaborating with the Erik Jorgensen laboratory here at the University of Utah, and will be exploring synapses in a model of retinal degeneration.  There is a nice writeup of the award on the Moran Eye Center website, here.

This is a wonderful opportunity to work with other colleagues that will be funded alongside us with this grant, including Uri Manor @manorlaboratory, Davi Bock @dddavi, Josh Vogelstein @neuro_data, Viren Jain @stardazed0, and others.  My thanks to Kristen Harris for heading up this initiative.

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

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.

Pathoconnectome Analysis of Müller Cells in Early Retinal Remodeling

We have a new manuscript out in Clinical Neurophysiology, An Update on Retinal Prostheses. PubMedDirect Link PDF here.

Authors: Rebecca L 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.

Abstract: Glia play important roles in neural function, including but not limited to amino acid recycling, ion homeostasis, glucose metabolism, and waste removal. During retinal degeneration and subsequent retinal remodeling, Müller cells (MCs) are the first cells to show metabolic and morphological alterations in response to stress. Metabolic alterations in MCs chaotically progress in retina undergoing photoreceptor degeneration; however, what relationship these alterations have with neuronal stress, synapse maintenance, or glia-glia interactions is currently unknown. The work described here reconstructs a MC from a pathoconnectome of early retinalremodeling retinalpathoconnectome 1 (RPC1) and explores relationships between MC structural and metabolic phenotypes in the context of neighboring neurons and glia. Here we find variations in intensity of osmication inter- and intracellularly, variation in small molecule metabolic content of MCs, as well as morphological alterations of glial endfeet. RPC1 provides a framework to analyze these relationships in early retinal remodeling through ultrastructural reconstructions of both neurons and glia. These reconstructions, informed by quantitative metabolite labeling via computational molecular phenotyping (CMP), allow us to evaluate neural-glial interactions in early retinal degeneration with unprecedented resolution and sensitivity.

 

 

Coupling architecture of the retinal Aii/ON cone bipolar cell network and alteration in degeneration

This poster was presented today, July 28th at the 2019 International Gap Junction Conference in Victoria, Canada by Crystal L. Sigulinsky, Rebecca L. PfeifferJames R. Anderson, Christopher Rapp, Jeebika Dahal, Jessica C Garcia, Jia-Hui Yang, Daniel P. Emrich, Hope Morrison, Kevin D. Rapp, Carl B. Watt, Mineo Kondo, Hiroko Terasaki, Robert E. Marc and Bryan W. Jones.

Almost full resolution version here.

Authors:

Crystal L Sigulinsky1, Rebecca L Pfeiffer1, James R. Anderson1, Christopher N. Rapp1, Jeebika Dahal1, Jessica C Garcia1, Jia-Hui Yang1, Daniel P. Emrich1, Hope Morrison1, Kevin D. Rapp1, Carl B. Watt1, Mineo Kondo2, Hiroko Terasaki3, Robert E. Marc1, Bryan W. Jones1
1Moran Eye Center/ Ophthalmology, University of Utah, Salt Lake City, Utah, United States; 2Mie University, Tsu, Japan; 3Nagoya University, Nagoya-shi, Japan;

ABSTRACT:

Background and aim:
Gap junctions are prevalent throughout the neural retina, with expression by every major neuronal class and at every level of signal processing. Yet, the functional roles and expressing cells/participating networks for many remain unknown. Spontaneous network spontaneous hyperactivity observed during retinal degeneration contributes to visual impairment and requires gap junctional coupling in the Aii amacrine cell/ON cone bipolar cell (CBC) network.  However, it remains unclear whether this hyperactivity reflects changes in the underlying circuitry or dysfunction of the normative circuitry. Here, we used connectomics-based mapping of retinal circuitry to 1) define the coupling architecture of the Aii/ON CBC network in healthy adult rabbit retina using connectome RC1 and 2) evaluate changes in coupling motifs in RPC1, a pathoconnectome from a rabbit retinal degeneration model.

Methods:

RC1 and RPC1 are connectomes built by automated transmission electron microscopy at ultrastructural (2 nm/pixel) resolution. RC1 is a 0.25 mm diameter volume of retina from a 13-month old, light adapted female Dutch Belted rabbit. RPC1 is a 0.07 mm diameter volume of degenerate retina from a transgenic P347L model of autosomal dominant retinitis pigmentosa (10-months old, male, New Zealand White background) presenting with ~50% rod loss. ON CBCs, Aii amacrine cells, and their coupling partners were annotated using the Viking application. Coupling motifs and features were explored with 3D rendering and network graph visualization. Gap junctions were validated by 0.25 nm resolution recapture with goniometric tilt when necessary.

Results:

Complete reconstruction of 37 ON CBCs in RC1 yielded 1339 gap junctions and revealed pervasive in- and cross-class coupling motifs among ON CBCs that produce complex network topologies within the coupled Aii network. Robust rulesets underlie class-specific coupling profiles with specificity defined beyond geometric opportunity. These coupling profiles enabled classification of all 145 ON CBCs contained within RC1 into 7 distinct classes. In RPC1, two ON CBC classes appear to retain their class-specific coupling profiles, accepting and rejecting specific combinations of Aii and ON CBC class partnerships. However, aberrant partnerships exist, including both loss of motifs and acquisition of novel ones.

Conclusions:

Gap junctions formed by ON CBCs are prominent network components, with specificity rivaling that of chemical synapses. These gap junctions not only subserve canonical signal transfer for night vision, but also extensive coupling within and across the parallel processing streams. Clearly aberrant morphological and synaptic changes exist in RPC1, including changes in the coupling specificity of both Aii and ON CBCs. Thus, circuit topology is altered prior to complete loss of rods, with substantial implications for therapeutic interventions for blinding diseases that depend upon the surviving retinal network.

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

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

Purpose
In mouse models of retinal degeneration, connexin36-containing gap junctions in the Aii amacrine cell network appear to mediate aberrant hyperactivity within the retina. However, it remains unclear whether this hyperactivity reflects changes in the underlying circuitry or dysfunction of the normative circuitry. Our connectomics-based mapping of retinal circuitry in rabbit Retinal Connectome 1 (RC1) has dramatically expanded the coupled Aii network. 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. This study examines the changes in these coupling motifs in RPC1, an ultrastructural retinal pathoconnectome from a rabbit model of retinitis pigmentosa.

Methods
RC1 and RPC1 are connectomes built by automated transmission electron microscopy at ultrastructural (2 nm/pixel) resolution. RC1 is a 0.25 mm diameter volume of retina from a 13 month old, light-adapted female Dutch Belted rabbit and serves as the healthy reference connectome. RPC1 is a 0.09 mm diameter volume of pathological retina from a 10 month old, male transgenic P347L model of autosomal dominant retinitis pigmentosa showing early phase 1 retinal remodeling, when rod photoreceptors are still present, but stressed. ON CBCs, Aii amacrine cells, and their coupling partners were annotated using the Viking application. Coupling motifs and features were explored with 3D rendering and graph visualization of connectivity. Gap junctions were validated by 0.25 nm resolution recapture with goniometric tilt when necessary.

Results
All major coupling motifs were observed. Several ON CBC classes retained their class-specific coupling profiles, accepting and rejecting specific combinations of Aii and ON CBC class partnerships. However, aberrant partnerships exist in the coupled network, including both loss of prominent motifs and acquisition of novel ones.

Conclusions
Clearly aberrant morphological and synaptic changes exist in RPC1, including changes in the coupling specificity and gap junction distributions of both Aii amacrine cells and ON CBCs. This indicates that the Aii/ON CBC circuit topology is already altered during early phase 1 remodeling, with substantial implications for therapeutic interventions for blinding diseases that depend upon the surviving retinal network in human patients.

Comparative Anatomy And Connectivity Of The AII Amacrine Cell In Mouse And Rabbit Retina

This abstract was presented today, April 8th at the 2019 Association for Research in Vision and Opthalmology (ARVO) meetings in Vancouver, Canada by Selena Wirthlin, Crystal L. SigulinskyJames R. Anderson, Daniel P. Emrich, Christopher Rapp, Jeebika Dahal, Rebecca L. Pfeiffer, Kevin D. Rapp, Jia-Hui Yang, Carl B. Watt, Robert E. Marc and Bryan W. Jones.

Full resolution version here.

Purpose
Mouse retina structurally differs from rabbit retina, as it is thicker and vascularized, while the rabbit retina is thinner and avascular. The implications of these differences on neuronal morphology and connectivity is not known. This project compares the morphology and connectivity of the Aii amacrine cell (AC) with ultrastructural precision in connectomes of mouse (RC2) and rabbit (RC1) retina.

Methods
RC1 and RC2 are connectomes built by automated transmission electron microscopy at ultrastructural (2 nm/pixel) resolution. RC1 and RC2 are 0.25mm diameter volumes of retina. RC1 is from a 13 month old, female Dutch Belted rabbit. RC2 is from a 5 month old female C57BL/6J mouse. The Viking application was used to annotate Aii ACs in both connectomes.

Results
Mouse Aii ACs are noticeably elongated to span the thicker inner plexiform layer (IPL) and have a prominent neck region. Lobular appendages of Aii ACs in both species extend thin stalks from the soma, neck and proximal arboreal dendrites in the OFF sublamina, predominantly forming reciprocal synapses with OFF cone bipolar cells (BCs). In rabbits, multiple arboreal dendrites emerge from the base of the neck, branch and travel obliquely through the ON sublamina, and form gap junctions with ON cone BCs, neighbor Aii ACs, and itself. They extend laterally at the base of the IPL, collecting ribbon input from rod BCs. In contrast, mouse arboreal dendrites stem from a single primary dendrite that branches as it travels vertically through the IPL without self-branch interaction, terminating at variable depths that align with the more broadly ramified axon terminals of rod BCs. Conventional synapse to gap junction ratios reveal greater output in the OFF vs ON layer in mouse compared to rabbit. Notably, mouse Aii ACs form gap junctions with the descending axons of ON cone BCs as they pass its soma, in contrast to rabbit, where gap junctions do not form at contacts proximal to ON cone BC axon terminals.

Conclusions
Lateral expansion of rabbit Aii ACs may be attributable to eccentricity. However, morphological differences appear to mediate greater output to the OFF versus ON pathway in mouse. Synaptic partners are currently being analyzed. Comparative anatomy connectomics is essential for understanding possible implications of retinal structure on neuronal morphology and connectivity that may underlie network differences between the mouse and rabbit retina.

Aii Amacrine Cell Connectivity in Degenerating Retina

This abstract was presented today, April 8th at the 2019 Association for Research in Vision and Opthalmology (ARVO) meetings in Vancouver, Canada by Jeebika Dahal, Rebecca L. Pfeiffer, Crystal L. Sigulinsky, James R. Anderson, Daniel P. Emrich, Hope Morrison, Jessica C. Garcia, 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
Aii amacrine cells (Aii ACs) function in mediating scotopic vision via connection of rod bipolar cells (Rod BCs) to cone bipolar cell pathways. The purpose of this project is to determine the effect of retinal degeneration (RD) on Aii AC networks. We explore this in a pathoconnectome of early RD (RPC1), using a connectome of healthy retina (RC1) as control. Cells in each volume are evaluated by comparison of morphology, synaptic connectivity, and eventually network analysis.

Methods
Tissue for RPC1 was collected from a 10 month old transgenic p347L rabbit model of autosomal dominant retinitis pigmentosa. RC1 was collected from a 13 month old Dutch-Belted rabbit, with no indications of degeneration. Tissue was fixed in a mixed aldehyde solution, before subsequent dehydration, osmication, and resin embedding. Volumes were sectioned at 70nm (RPC1) and 90nm (RC1) and placed on formvar grids. 1 section was reserved from every 30 TEM sections for computational molecular phenotyping where it was placed on a slide and probed for small molecules or proteins. TEM sections were captured at 2.18nm/px using SerialEM software on a JEOL JEM-1400 TEM. The RC1 volume has a diameter of 250µm and RPC1 has a diameter of 90µm. Both volumes were analyzed using the Viking software suite.

Results
In this study, Aii ACs from RPC1 were compared to RC1. Initial results indicate no distinct difference in the morphology other than arbor size, which are likely due to eccentricity differences between volumes. However, in RPC1, we observe multiple instances of Aii AC coupling with Rod BCs in the ON region of the IPL. In contrast, Rod BCs never form gap junctions in healthy retina.

Conclusions
Coupling between Aii ACs and Rod BCs in RPC1 is a unique change in retinal network topology occurring in early RD. Further exploration of network changes as a response to RD is warranted, as many therapeutic interventions currently in development rely upon maintenance of inner retinal circuitry. Prior research demonstrates Rod BCs extend dendrites towards cones and change their receptor expression as rods degenerate. Therefore, knowing the network changes involving Aii ACs and their associations with bipolar cells is crucial to understanding how photoreceptor degeneration affects inner retinal visual processing.

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

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.