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Metabolic, Excitation and Functional Mapping of Diabetic Retinopathy

This abstract was presented today, Monday, April 30th at the 2018 Association for Research in Vision and Opthalmology (ARVO) meetings in Honolulu, Hawaii by Felix R. Vazquez-Chona, Tam T.T. Phuong, Oleg Yarishkin, Bryan W. Jones, and David Krizaj

Loss of vision in diabetic retinopathy is associated with extensive shifts in retinal metabolic and synaptic function yet the general principles that govern the metabolic remodeling remain unknown. To define the metabolic signature in hyperglycemic retina we took advantage of in situ metabolomics, excitation mapping and gene knockdown. Specifically, we investigated whether manipulation of the swelling-activated calcium-permeable TRPV4 (transient receptor potential isoform 4) channel contributes to the metabolic program of the degenerating neurogliovascular subunit in diabetic mice.

Type I diabetes in wild type (WT) and TRPV4-/- mice was induced with streptozotocin (STZ). We visualized glutamate (NMDA)-gated excitation and glucose transport using the organic cation agmatine (AGB2+) and the glucose analog glucosamine (GCN). Retinas were fixed in glutaraldehyde, sectioned, and incubated with antibodies targeting GCN and AGB. Cell classification and metabolic status were interrogated using Computational Metabolic Profiling (CMP) and probes against ADP, alanine, arginine, aspartate, citrulline, GABA, glutamate, glycine, glutathione, glutamine, isoleucine, taurine, glutamine synthetase, CRALBP, GFAP, and tomato lectin.

Amacrine and ganglion cells in control retinas responded to NMDA activation with a large elevations in AGB and GCN signals. Diabetic amacrine cells maintained a robust dynamic range of AGB and GCN signals which however were markedly diminished in RGCs. Metabolomic maps of diabetic WT retinas showed that the outer retina remains metabolically quiescent whereas the ganglion cell layer displayed cells with lower glutamate and GABA signals. Diabetic TRPV4-deficient retinas displayed metabolomic, excitation, and glucose transport maps that were comparable to control retinas. ERG analysis showed modest STZ-induced changes in scotopic a- and b-waves of WT and KO eyes.

Our preliminary electrophysiological and metabolomic findings suggest that STZ-induced diabetes spares the inner retina but alters amacrine-ganglion cell signaling, the neurogliovascular unit organization together with RGC metabolism. TRPV4 inactivation partially rescues the metabolic, excitation, physiologic phenotypes imposed by hyperglycemia. These results suggest that ambient sensing through polymodal TRP channels links retinal neuronal, glial and endothelial signaling to cellular metabolism and visual function.

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


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.

Robust Segmentation based Tracking using an Adaptive Wrapper for Inducing Priors


We have published another manuscript, Robust Segmentation based Tracking using an Adaptive Wrapper for Inducing Priors.  This manuscript describes the work on adaptive tracing and proposes an algorithm that adapts a generic tracing algorithm to an application of interest.  In our specific case, it is boundaries of cells in high frequency space in transmission electron microscopy images.  But the approach in this paper is applicable to biological, medical, remote sensing and surveillance data as well utilizing priors specific to the application.  The co-authors on the paper are: Vignesh JagadeeshJames Anderson, Bryan W. JonesRobert E. MarcSteven K Fisher and B.S Manjunath.