Category Archives: Abstracts

In Situ Metabolomic Signatures Of Neuroprotection, Apoptosis, And Microglial Phagocytosis

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This abstract was presented July 1st at the 11th International Converence of the Metabolomics Society at the University of California, Davis by Felix Vazquez-Chona, Drew Ferrell, Bryan W. Jones and Robert E. Marc.

 

Metabolic dysregulation is an early hallmark of neurodegenerative diseases including Alzheimer’s disease and age-related macular degeneration. Mapping metabolic adaptation with cellular resolution and tissue- wide context is crucial to define networks regulating neuronal survival, cell death progression, and immune cell response.

Computational Molecular Phenotyping (CMP) explores the amine metabolome (amino acids and amines). Technically, CMP metabolomics combines amine metabolite trapping, ultrathin microscopy (50-200 nm), immunodetection, pattern recognition, and clustering algorithms. Here we mapped the in situ distribution of over 30 core amine metabolites in retinal cells challenged by light-induced oxidative stress. Metabolomic profiles were phenotyped using ultrastructural, biochemical, and proteomic indices of oxidative stress.

CMP enabled precise visualization of >30 metabolites in every retinal cell. CMP resolved and phenotyped metabolomic profiles to specific degeneration and microglial functional states in the light-damaged retina. Cone photoreceptor survival correlated with enhanced antioxidant glutathione content. Rod photoreceptor apoptosis coincided with rapid depletion of organic osmolytes followed by nuclear import of cationic arginine metabolites. Delay in cell death increased necrosis and DNA damage-induced apoptosis. Microglial chemotaxis enhanced distinct signatures of glutamate and glutathione metabolism; whereas, phagocytosis coinduced classic (M1) and alternative (M2) arginine metabolites of macrophage activation.

CMP discovers and phenotypes cell classes, tracks cell state, and maps disease with single-cell resolution in any tissue or organism.

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

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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

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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).

Metabolic Changes Associated With Müller Cells In A Transgenic Rabbit Model Of Retinal Degeneration

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Retina-RLP

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).

Constructive Retinal Plasticity After Selective Ablation of the Photoreceptors

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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.

Purpose: In the rabbit retina there is evidence of constructive retinal plasticity in response to focal ablation of a small patch of the photoreceptor layer by laser photocoagulation. After a two-month healing period, healthy photoreceptors migrate inwards filling the damaged area and restoring visual sensitivity to the lesion site. We investigated the integrity and function of the neural populations above the lesion, whether the migrating photoreceptors formed new connections with deafferented bipolar cells, and to what degree the new function resembled normal retinal function.

Methods: Retinal photocoagulation lesions of Moderate and Barely Visible clinical grades were produced in rabbits with a 532-nm laser, using beam diameter of 200 and 400 μm. Retinal ganglion cell (RGC) responses to spatio-temporal white noise stimulus were recorded on a 512-electrode array. Inner retinal neuron cell types were identified using Computational Molecular Phenotyping (CMP). Light evoked activity of the inner retinal neurons was measured through 1-amino-4-guanidobutane (AGB) labeling. Synaptic structure between photoreceptors and bipolar cells was characterized through transmission electron microscopy (TEM) imaging.

Results: The lesioned areas of the retina, after a two-month healing period, regained visual sensitivity. There was no significant difference between the response kinetics of RGCs with receptive fields covering the lesioned area and RGCs with receptive fields unaffected by the lesion. Furthermore, the average receptive field sizes of RGCs covering the lesion were consistent with the average receptive field sizes of RGCs unaffected by the lesion. CMP showed that all major inner retinal neuron cell types are present above both acute and healed lesions. Light evoked activity in the retina, as measured by AGB concentration levels, was diminished in the acute lesion but returned to within 10% of normal after two months. TEM images showed normal photoreceptor synaptic structure inside the healed lesion area.

Conclusions: Migrating photoreceptors establish new functional connectivity to deafferented bipolar cells and have normal synaptic structure. The new circuitry results in spatial and temporal properties of the RGC responses that resemble those of the healthy retina. In summary, the rewiring restores normal visual response in the lesioned area, indicating constructive retinal plasticity.

Support: Burroughs Wellcome Fund Career Award at the Scientific Interface; the Pew Charitable Trusts Scholarship in the Biomedical Sciences (A.S.), RPB CDA, Thome Foundation (BWJ), NIH EY02576, NIH EY015128, NSF 0941717, NIH EY014800 Vision Core (R.M.); NIH 5R01EY18608, AFOSR FA9550- 10-1-0503, DoD W81XWH-12-10575, Stanford University Bio-X (D.P.), Edward N. and Della L. Thome Memorial Foundation grant for Age-Related Macular Degeneration Research (BWJ) RPB unrestricted award (Moran Eye Center)

Retinal Metabolic Response to Cigarette Smoke

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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.

Purpose:  Smoking is the single largest risk factor for age-related macular degeneration, aside from age. Several of the main genetic risk factors for AMD are polymorphisms occurring in complement genes involved in the alternative, classical and common terminal pathways. To better understand the metabolic impact of smoking on the retina, we used computational molecular phenotyping (CMP) and examined the effects of cigarette smoke on wild type (wt) retinas and mice in which either the alternative pathway (complement factor B, CfB) or the common terminal pathway (complement component 3, C3) was removed.

Methods:  Mice were exposed to either cigarette smoke or filtered air. Cigarette smoke (CS) was generated using an automated cigarette-smoking machine (Model TE-10, Teague Enterprises, Davis, CA) by burning 3R4F reference cigarettes (2.45 mg nicotine per cigarette; purchased from the Tobacco Health Research Institute, University of Kentucky, Lexington, KY). Mice were exposed to CS for 6 hours/day, 5 days/week for 6 months. Age matched room filtered air exposed mice were used as controls. Eyes were enucleated immediately post-mortem, fixed in 1% paraformaldehyde, 2.5% glutaraldehyde, dehydrated in graded methanols, embedded in eponates and histologically analyzed with CMP.

Results:  Alterations in retinal small molecule signatures from mice exposed to cigarette smoke were observed compared to retinas from non-smoked mice in wt, CfB and C3 knockout mice. Signal changes with arginine, glutamine and glutathione progressively increased in the retinas of smoked exposed wt, CfB and C3 knockout mice, indicating increased response profiles to cell stress. Both Müller cells and photoreceptors of wt smoked retinas demonstrated changes relative to non- smoked retinas.

Conclusions:  Arginine, glutamine and glutathione, amino acids known to be involved in cellular stress responses, were increased in retinal neurons and glial cells upon smoke exposure. Eliminating essential components of the complement system, a cascade required for the maintenance of the immune privilege of the eye, appears to exacerbate responses to cigarette smoke in oxidative damage response related pathways. Understanding complement-dependent alterations in the eye will aid in our understanding of AMD pathology and may open new avenues for novel treatment strategies.

Support:  RPB CDA (BWJ), Thome AMD Grant (BWJ), NIH EY02576 (RM), NIH EY015128 (RM), NSF 0941717 (RM), NIH EY014800 Vision Core (RM), NIH EY019320 (BR), VA merit award RX000444 (BR), grant to MUSC from RPB