Category Archives: Connectomics

Heterocellular Coupling Between Amacrine Cells and Ganglion Cells

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.

Rod Bipolar Cell Networks in Early Retinal Remodeling

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.

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

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.


SEM vs. TEM is a tradeoff of convenience, resolution, cost and speed. The very physics of SEM signal integration means that the fundamental acquisition time for large canonical volume datasets are incompatible with 5 year grant cycles. SEM based approaches can potentially rival TEM, but dwell time/pixel increases logarithmically with resolution.

To give you some idea for the resolution differences at routine capture speeds, both of these above images capture a region within the inner plexiform layer of retina, looking at bipolar cell terminals. The TEM image was captured at a standard operating resolution of 2nm/pixel. The SEM image was captured at 16nm/pixel. You cannot see any gap junctions that might be present in the SEM image and you can only infer or guess at synaptic ribbons. And look at the texture!

You *can* get better resolution with SEM, but as I said before, the capture time increases logarithmically. To accomplish what we perform in 8-10 hours with a TEM, would take 108-115 hours on a current, cutting edge multi beam SEM. There are many other advantages of TEM including the ability to capture higher resolution images faster, be able to re-image in goniometric tilt series, be able to integrate molecular markers inside connectome volumes, and a TEM is about 1/3rd the cost of an SEM. Also, SEM images tend to be texturally poor as they are made from capturing electron backscatter of surfaces rather than made by projection of electrons through a small volume, and there is tremendous value in the texture of ultrastructural images. Ergo, this is why we use TEM.

This is not to say that SEM is not a great tool. It is just not the best tool for large scale connectomics where you have to have the resolution to capture all synapses and gap junctions, over large areas. For smaller volumes that do not require a canonical sampling of cell classes, SEM is absolutely an appropriate tool.

This content was originally published on Jonesblog.

Nornir listed on NITRC

Nornir is now registered with NeuroImaging Tools & Resources Collaboratory (NITRC).

Nornir’s takes large sets of overlapping images in 2D and produces registered (a.k.a. aligned) 2D mosaics and 3D volumes of any size and scale. Registered slices may be exported as a single large images or viewed/annoted with our Viking viewer.

Nornir has been used successfully on transmission electron microscopy, scanning electron microscopy images, and light microscopy images. Nornir supports interleaving different imaging methods into the same volume. Support for SerialEM, Objective Imaging, and Digital Micrograph (DM4) raw data is available. Adding formats is not complicated and the author will consider requests.

Nornir runs on fairly humble hardware for the task. A 32-core 64GB Xeon system built a ~60 TB 250um diameter 2.12nm/pixel volume from roughly 1400 slices. Nornir works incrementally, only updating data that has changed.

Installation is fairly simple and primarily uses Python’s PIP installer.

For further information:

A Pathoconnectome of Early Retinal Remodeling

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.

Circuit Remodeling In Retinal Degeneration

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.

The Rod-Cone Crossover Connectome of Mammalian Bipolar Cells

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

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. Brown, Rebecca L. Pfeiffer, Crystal Sigulinsky, Felix Vazquez-Chona, Daniel Emrich, Bryan W. Jones, Robert E. Marc
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

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.