We have a new Webvision chapter on Retinal Degeneration, Remodeling and Plasticity! Check it out here.
This abstract was presented today at the 2015 Association for Research in Vision and Opthalmology (ARVO) meetings in Denver, Colorado by Rebecca L. Pfeiffer, Bryan W. Jones and Robert E. Marc.
Purpose: Müller cells (MCs) play a critical role in glutamate (E) metabolism and carbon skeleton cycling in retina. MCs demonstrate changes in metabolism and morphology during retinal degeneration. The timing, extent, regulation, and impacts of these changes are not yet known. We evaluated metabolic phenotypes of MCs and evaluated their capacity to transport glutamate during degeneration.
Methods: Retinas were harvested from wild-type (WT) and rhodopsin Tg P347L rabbits, divided into chips mounted on filters, and incubated in Ames medium with 5 mM D-aspartate (D-Asp), D-glutamate (D-Glu), or D-glutamine (D-Gln) for 10 min at 35 deg to explore transport and metabolism. Chips were fixed in mixed aldehydes and resin embedded for computational molecular phenotyping (CMP) of a range of L- and D-amino acid markers and selected proteins including glutamine synthetase (GS) (J Comp Neurol. 464:1, 2003).
Results: CMP revealed wide variations in metabolite levels across individual MCs from Tg P347L retinas, generating chaotic patterns. GS decreased significantly while glutamine levels (Q) increased, although to varying degrees. Remarkably, E levels were variable and much higher in some MCs than normal, but did not correlate (inversely) with GS levels. Transport experiments using D-Glu, D-Asp, and D-Gln showed that alterations in MC metabolites are not the product of defective transporters, in contrast to previous reports. These results are also inconsistent with conventional models of GS-based E-Q metabolism and microenvironmental regulation of MC phenotypes.
Conclusions: These observations suggest three conclusions. (1) Although degeneration of the retina is certainly the trigger, MC phenotype changes are not a coherent response to the surrounding microenvironment but are, rather, uncoordinated individual MC responses. (2) Although GS is accepted as the primary enzyme responsible for the conversion of E to Q in the normal retina, alternative pathways appear unmasked in the degenerate state. (3) It has been previously hypothesized that MCs in retinal degenerations exhibit deficient E transport. Our experiments show no transport deficiency. This indicates that chaotic metabolite levels emerge from changes in individual MC metabolic processing.
Our colleague and Director of Research at the University of Utah‘s Moran Eye Center, Robert E. Marc, Ph.D. has been named by the International Society for Eye Research as a recipient of the Paul Kayser International Award in Retina Research. The award will be presented to Dr. Marc during the 2014 ISER Biennial Meeting of the Society for Eye Research in San Francisco, California on Thursday, July 24th, 2014.
The Paul Kayser International Award in Retina Research was created by the Directors of Retina Research Foundation and endowed by the Trustees of The Kayser Foundation to honor and perpetuate the memory of long-time friend and dedicated benefactor of RRF, Paul Kayser. Through this award both organizations are demonstrating the conviction they shared with Mr. Kayser that blindness caused by retinal disease is a global concern and must be addressed accordingly. It is thus the purpose of this award to foster greater awareness of the need for intensive study of the retina, its role in the visual process, and the retinal diseases that threaten and/or destroy eyesight by recognizing outstanding achievement and sustaining meritorious scientific investigations worldwide.
Dr. Marc was chosen as the recipient of this award for his lifetime body of work in retinal research, discovering the structure and function of the retina through novel technologies and approaches that have pushed our understanding of the retina forward.
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).
This abstract was presented today at the 2014 Association for Research in Vision and Opthalmology (ARVO) meetings in Orlando, Florida by Rebecca L. Pfeiffer, Bryan W. Jones and Robert E. Marc.
Purpose: Müller cells play a central role in retinal metabolism via the glutamate cycle. During retinal degeneration Müller cells are among the first to demonstrate changes, reflected in alterations of metabolic signatures and morphology. The timing, extent and regulation of these changes is not fully characterized. To address this issue, we evaluated Müller cell metabolic phenotypes at multiple stages of retinal remodeling.
Methods: Samples were collected post-mortem from both WT and P347L rabbits. The retinas were then divided into fragments, fixed in buffered aldehydes, and embedded in epoxy resins. Tissues were sectioned at 200nm followed by classification with computational molecular phenotyping (CMP) using an array of small and macromolecular signatures (aspartate (D), glutamate (E), glycine (G), glutamine (Q), glutathione (J), GABA (yy), taurine (T), CRALBP, Glutamine Synthetase (GS), and GFAP). Levels of amino acid or protein were quantified by selecting a region of interest either within the Müller cell population or surrounding neurons and evaluating the intensity of the signal within that region.
Results: CMP reveals overall decreases in GS levels over the course of degeneration. Of notable importance, we saw that in regions of near complete photoreceptor loss neighboring Müller cells may express independent variation in metabolic signatures of E, Q, and GS. Also observed in these Müller cells, ratios of GS:E and GS:Q are not consistent with the ratios seen in WT retina. These results are inconsistent with the current models of both E to Q metabolism and microenvironment regulation of Müller cell phenotypes.
Conclusions: These observations indicate two conclusions. First, although the degenerate state of the retina is the likely trigger inducing Müller cells to express altered metabolic signatures, the rate at which the metabolic state changes is not purely a product of the surrounding environment, but also a stochastic change within individual Müller cells. Second, although it is commonly accepted that GS is the primary enzyme which converts Q to E as part of the glutamate cycle, in degenerate retina alternative pathways may be utilized following decrease in GS.
Support: NIH EY02576 (RM), NIH EY015128 (RM), NSF 0941717 (RM), NIH EY014800 Vision Core (RM), RPB CDA (BWJ), Thome AMD Grant (BWJ).
This abstract was presented today at the Association for Research in Vision and Opthalmology (ARVO) meetings in Seattle, Washington by Alexandra D. Butler, William D. Ferrell, Alex Woodell, Carl Atkinson, Baerbel Rohrer, Robert E. Marc and Bryan W. Jones. Continue reading “Retinal Metabolic Response to Cigarette Smoke”
This abstract was presented today at the Association for Research in Vision and Opthalmology (ARVO) meetings in Seattle, Washington by Corinne N. Beier, Bryan W. Jones, Philip Huie, Yannis M. Paulus, Daniel Lavinsky, Loh-Shan B. Leung, Hiroyuki Nomoto, Robert E. Marc, Daniel V. Palanker, and Alexander Sher. Continue reading “Constructive Retinal Plasticity After Selective Ablation of the Photoreceptors”
This abstract was presented today at the Association for Research in Vision and Opthalmology (ARVO) meetings in Seattle, Washington by Crystal L. Sigulinsky, J. Scott Lauritzen, John V. Hoang, Carl B. Watt, Bryan W. Jones, James R. Anderson, Shoeb Mohammed and Robert E. Marc. Continue reading “Sparse Network Principles of GABAergic Amacrine Cell Heterocellular Coupling”
This abstract was presented today at the Association for Research in Vision and Opthalmology (ARVO) meetings in Seattle, Washington by J. Scott Lauritzen, John V. Hoang, Crystal Sigulinsky, Bryan W. Jones, James R. Anderson, Carl B. Watt, Shoeb Mohammed and Robert E. Marc. Continue reading “Tiered Cross-Class Bipolar Cell Gap Junctional Coupling in the Rabbit Retina”
This abstract was presented today at the Association for Research in Vision and Opthalmology (ARVO) meetings in Seattle, Washington by Robert E. Marc, Felix R. Vazquez-Chona, John V. Hoang, Crystal Sigulinsky, Carl B. Watt, Bryan W. Jones, James R. Anderson and J. Scott Lauritzen. Continue reading “Pure Feedforward Amacrine Cells”
This abstract was presented today at the Association for Research in Vision and Opthalmology (ARVO) meetings in Seattle, Washington by Steven J. Fliesler, Christopher C. Goulah, W. Drew Ferrell, Robert E. Marc and Bryan W. Jones. Continue reading “Retinal Remodeling in a Rat Model of the Smith-Lemli-Opitz Syndrome (SLOS)”
There has been quite a bit of discussion of connectomes in the last while with President Obama’s new BRAIN initiative. It is important to consider some of the requirements of obtaining a true synapse level wiring map in the brain as many are articulating from this initiative. While there are new technologies that will be required to undertake this initiative for mapping the entire brain, the NIH NEI has been funding an ongoing project to study retinal circuitry which guides the community in how to approach a true synapse level map of the nervous system.
An example of this work in Current Opinion in Neurobiology titled Building Retinal Connectomes is a review that illustrates the importance of having a complete network graph of connectivities in the retina and by extension other neural systems. Complete network graphs are what will be required to understand how retinal systems (and any neural system) is constructed. Even though the retinal community understands how retinas are wired in broad strokes, the precise, fine details are critical and elucidating them requires a new level of complete annotation derived from advances in light and ultrastructural imaging, data management, navigation and validation.
This paper in the Journal of Comparative Neurology by J. Scott Lauritzen, James R. Anderson, Bryan W. Jones, Carl B. Watt, Shoeb Mohammed, John V. Hoang and Robert E. Marc is another effort out of the Marc Laboratory For Connectomics that continues to define complete neural circuits to completeness.
This paper is another elucidation of data from the first Rabbit Retinal Connectome volume (RC1) that reveals that the division between the ON and the OFF inner plexiform layer (IPL) is not structurally absolute. ON cone bipolar cells make noncanonical axonal synapses onto specific targets and receive amacrine cell synapses in the nominal OFF layer, creating novel motifs, including inhibitory crossover networks. Automated transmission electron microscopic imaging, molecular tagging, tracing, and rendering of ∼400 bipolar cells reveals axonal ribbons in 36% of ON cone bipolar cells, throughout the OFF IPL. The targets include γ-aminobutyrate (GABA)-positive amacrine cells (γACs), glycine-positive amacrine cells (GACs), and ganglion cells. Most ON cone bipolar cell axonal contacts target GACs driven by OFF cone bipolar cells, forming new architectures for generating ON–OFF amacrine cells. Many of these ON–OFF GACs target ON cone bipolar cell axons, ON γACs, and/or ON–OFF ganglion cells, representing widespread mechanisms for OFF to ON crossover inhibition. Other targets include OFF γACs presynaptic to OFF bipolar cells, forming γAC-mediated crossover motifs. ON cone bipolar cell axonal ribbons drive bistratified ON–OFF ganglion cells in the OFF layer and provide ON drive to polarity-appropriate targets such as bistratified diving ganglion cells (bsdGCs). The targeting precision of ON cone bipolar cell axonal synapses shows that this drive incidence is necessarily a joint distribution of cone bipolar cell axonal frequency and target cell trajectories through a given volume of the OFF layer. Such joint distribution sampling is likely common when targets are sparser than sources and when sources are coupled, as are ON cone bipolar cells.
Figure Above: The first Rabbit Retinal Connectome volume (RC1), constructed via automated transmission electron microscopy (ATEM) and computational molecular phenotyping (CMP), spans the mid-inner nuclear layer (INL) at section 001 to the ganglion cell layer (GCL) at section 371, shown in a mirror image below. RC1 is a short cylinder ≈ 250 μm in diameter and ≈ 30 μm high containing 341 ATEM sections and 11 intercalated CMP sections. The cylinder is capped at top and bottom with 10-section CMP series allowing molecular segmentation of cells, and an activity marker, 1-amino-4-guanidobutane (AGB), to mark cells differentially stimulated via glutamatergic synapses. ATEM section 001 is a horizontal plane section through the INL visualized with GABA.glycine.glutamate → red.green.blue transparency mapping and a dark gold alpha channel (ANDed taurine + glutamine channels). ATEM section 371 is a horizontal plane section through the GCL visualized with GABA.AGB.glutamate → red.green.blue transparency mapping.
We at Webvision would like to wish you all a very happy holiday season and a happy New Year in 2013. Continuing on the theme from last years holiday season wishes, we have for you a colorful holiday image titled “Connectomics, First Light” from Robert E. Marc utilizing data generated by Scott Lauritzen, Crystal Sigulinsky and John Vo Hoang.
What we are seeing is a necklace of coupled retinal bipolar cells linked by sparse suboptical gap junctions, fundamentally new circuitry for retinal processing. There is much more to this story, but we’ll wait until the publication. For now, consider these data generated from the Connectomics project in the Marclab at the Moran Eye Center to be a colorful holiday image that represents our best wishes to you.
This poster and presentation was presented today at the Federation of American Societies For Experimental Biology (FASEB) meeting in Steamboat Springs, Colorado by J. Scott Lauritzen, James R. Anderson, Carl B. Watt, Bryan W. Jones and Robert E. Marc. A large size pdf of the poster can be downloaded here.