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.
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.
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.
This abstract was presented at the 2014 Society for Neuroscience meeting in Washington D.C. by J. Scott Lauritzen, Crystal L. Sigulinsky, Danny P. Emrich, Joshua M. Dudleston, Noah T. Nelson, Rebecca L. Pfeiffer, Nathan R. Sherbotie, John V. Hoang, Jefferson R. Brown, Carl B. Watt, James R. Anderson, Bryan W. Jones and Robert E. Marc.
Purpose: Despite large-scale efforts aimed at mapping the mammalian nervous system, the entire synaptic cohort of a single mammalian neuron of any class has never been mapped. To this end we reconstructed all chemical and electrical synaptic partners of a single ON cone bipolar cell (ON CBC) in the inner plexiform layer (IPL) of the rabbit retina. We then searched all members of the same cell class for repeating network motifs and explored postsynaptic cell sampling topologies from this bipolar cell (BC).
Methods: Cells in retinal connectome 1 (RC1) were annotated with Viking viewer, and explored via graph visualization of connectivity and 3D rendering (Anderson et al., 2011 J Microscopy). Small molecule signals in RC1, e.g. GABA, glycine, and L-glutamate, combined with morphological reconstruction and connectivity analysis allow robust cell classification. The default resolution of RC1 is 2.18nm/pixel, however goniometric recapture at 0.273 nm/pixel was performed as needed for synapse verification.
Results: ON CBC 593 is one of 20 BCs of this class in RC1, the axonal arbors of which tile with gap junctions between nearest neighbors at their distal axonal tips. ON CBC 593 contains 194 ribbons, 274 postsynaptic densities, 20 gap junctions, and 66 conventional synapses, for a total of 554 synaptic connections. Twenty ganglion cells sample the glutamatergic output. ON CBC 593 is presynaptic to 262 amacrine cell (AC) processes, and is postsynaptic to 228 AC processes. Of these, 33% form reciprocal connections. We approximate that ON CBC 593 forms synapses with 50 distinct ACs. ON CBC 593 is routinely pre- and postsynaptic to within-class, cross-class, feedback, and feedforward inhibition motifs, including 1 instance of OFF-ON crossover inhibition. ON CBC 593 forms 12 gap junctions with at least 2 AII ACs, 7 with 5 ON CBCs, and 1 with itself. We searched for repeating network motifs across all ON CBCs of this class in RC1. Thus far, 80% of these form in-class inhibitory motifs, and 75% form cross-class inhibitory motifs. All ACs and GCs discovered to contact multiple branches of ON CBC 593 form synapses on every branch.
Conclusions: An individual bipolar cell is inherently multi-kinetic, receiving inhibition driven by all ON CBC classes, sharing these signals via gap junctions with ON CBCs of the same class, and driving inhibition of all ON CBC classes. This constitutes a substrate for multi-channel coordination throughout the IPL, and predicts multi-kinetic BC responses. The results establish a normative framework against which members of the same and different classes may be compared, and foster interpretation of BC physiological behavior under different stimulus regimes.
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.
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).