Tag Archives: Crystal L. Sigulinsky

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.

Pathoconnectome Analysis of Müller Cells in Early Retinal Remodeling

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.

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.

Ultrastructural Connectomics Reveals The Entire Chemical And Electrical Synaptic Cohort Of An ON Cone Bipolar Cell In The Inner Plexiform Layer Of The Rabbit Retina

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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. WattJames 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.

Ultrastructural Reconstruction of ON Cone Bipolar Cell Projective Fields In The Innter Plexiform Layer of The Rabbit Retina

retina reconstruction

This abstract was presented at the 2014 FASEB Summer Research Conference in Saxtons River, Vermont by J. Scott Lauritzen, Crystal L. Sigulinsky, Noah T. Nelson, Nathan R. Sherbotie, Danny P. Emrich, Rebecca L. Pfeiffer, Jefferson R. Brown, John V. Hoang, Joshua M. Dudleston, Carl B. Watt, Kevin Rapp, Marguerite V. Shaw, Jia-Hui Yang, James R. Anderson, Bryan W. Jones and Robert E. Marc.

Purpose: Functional mapping in tiger salamander shows that bipolar cell (BC) projective fields far exceed their axonal fields, and directly implicates wide-field GABAergic amacrine cells (wf γACs) and gap junctions (Asari & Meister, 2014). Strikingly, single BCs exert differential effects on functionally distinct ganglion cells (GCs), likely achieved by privatized amacrine cell (AC) presynaptic inhibition to specific BC-GC synaptic pairs (Asari & Meister, 2012). To address whether BC projective fields in the mammal are equally broad, wf γAC- and gap junction-dependent, and GC type unselective, we reconstructed all electrical and chemical synaptic partners of a single ON cone BC in the inner plexiform layer of the rabbit retina, and searched BC-GC synaptic pairs for differential synaptic inhibition.

Methods: Cells in retinal connectome 1 (RC1) were annotated with Viking viewer, and explored via connectivity visualizations and 3D rendering (Anderson et al., 2011). Small molecule signals embedded in RC1, e.g. GABA, glycine, and L-glutamate, combined with morphological reconstruction and connectivity analysis allow robust cell classification. We used the MacNeil et al. (2004) rabbit BC classification scheme.

Results: CBb5w 593 is one of 20 ON cone BCs of this class in RC1. This CBb5w is presynaptic to 17 distinct GCs and 262 AC processes, and postsynaptic to 228 AC processes. The majority of these ACs are wf γACs. We estimate this BC forms synapses with 50 unique ACs. Asari & Meister (2014) found that single bipolar cell projective fields range up to 1 mm, far beyond a BC axonal field, and differentially drive multiple classes of GC. We discovered BC-BC within- and cross-class coupling and lateral inhibition that construct sign-conserving and sign-inverting projective fields to many distinct ganglion cell classes across the entire 0.25 mm diameter of RC1, much greater than a 60 µm BC axonal field. Cross-class projections access a broader set of GCs than expected from in-class projections alone. The BC-BC coupling is independent of BC-AII AC coupling. 94% of the CBb5w 593 BC-GC synaptic pairs receive feedback inhibition within the varicosity of the ribbon, but the number of feedback synapses is highly variable (coefficient of variation = 0.81). 35% of the BC-GC pairs receive feedforward inhibition within 2 microns of the postsynaptic density.

Conclusions: Mammalian BCs use novel cross-class topologies to distribute signals to a wide range of GCs and establish projective fields similar to those discovered in non-mammalian species. BC-BC within- and cross-class coupling and lateral inhibition via wf γACs establish sign-conserving and sign-inverting projective fields, respectively, up to 1 mm diameters. BC-GC synaptic pairs overwhelmingly employ feedback vs. feedforward inhibition to modulate signaling, and the numbers of feedback synapses are highly variable across these pairs, accounting for privatized and differential GC responses to the same BC drive.

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

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. PfeifferJames 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).