Bryan W. Jones

Neural remodeling in retinal degeneration

Mammalian retinal degenerations initiated by gene defects in rods, cones or the retinal pigmented epithelium (RPE) often trigger loss of the sensory retina, effectively leaving the neural retina deafferented. The neural retina responds to this challenge by remodeling, first by subtle changes in neuronal structure and later by large-scale reorganization. Retinal degenerations in the mammalian retina generally progress through three phases. Phase 1 initiates with expression of a primary insult, followed by phase 2 photoreceptor death that ablates the sensory retina via initial photoreceptor stress, phenotype deconstruction, irreversible stress and cell death, including bystander effects or loss of trophic support. The loss of cones heralds phase 3: a protracted period of global remodeling of the remnant neural retina. Remodeling resembles the responses of many CNS assemblies to deafferentation or trauma, and includes neuronal cell death, neuronal and glial migration, elaboration of new neurites and synapses, rewiring of retinal circuits, glial hypertrophy and the evolution of a fibrotic glial seal that isolates the remnant neural retina from the surviving RPE and choroid. In early phase 2, stressed photoreceptors sprout anomalous neurites that often reach the inner plexiform and ganglion cell layers. As death of rods and cones progresses, bipolar and horizontal cells are deafferented and retract most of their dendrites. Horizontal cells develop anomalous axonal processes and dendritic stalks that enter the inner plexiform layer. Dendrite truncation in rod bipolar cells is accompanied by revision of their macromolecular phenotype, including the loss of functioning mGluR6 transduction. After ablation of the sensory retina, Müller cells increase intermediate filament synthesis, forming a dense fibrotic layer in the remnant subretinal space. This layer invests the remnant retina and seals it from access via the choroidal route. Evidence of bipolar cell death begins in phase 1 or 2 in some animal models, but depletion of all neuronal classes is evident in phase 3. As remodeling progresses over months and years, more neurons are lost and patches of the ganglion cell layer can become depleted. Some survivor neurons of all classes elaborate new neurites, many of which form fascicles that travel hundreds of microns through the retina, often beneath the distal glial seal. These and other processes form new synaptic microneuromas in the remnant inner nuclear layer as well as cryptic connections throughout the retina. Remodeling activity peaks at mid-phase 3, where neuronal somas actively migrate on glial surfaces. Some amacrine and bipolar cells move into the former ganglion cell layer while other amacrine cells are everted through the inner nuclear layer to the glial seal. Remodeled retinas engage in anomalous self-signaling via rewired circuits that might not support vision even if they could be driven anew by cellular or bionic agents. We propose that survivor neurons actively seek excitation as sources of homeostatic Ca(2+) fluxes. In late phase 3, neuron loss continues and the retina becomes increasingly glial in composition. Retinal remodeling is not plasticity, but represents the invocation of mechanisms resembling developmental and CNS plasticities. Together, neuronal remodeling and the formation of the glial seal may abrogate many cellular and bionic rescue strategies. However, survivor neurons appear to be stable, healthy, active cells and given the evidence of their reactivity to deafferentation, it may be possible to influence their emergent rewiring and migration habits.

Retinal remodeling triggered by photoreceptor degenerations

Many photoreceptor degenerations initially affect rods, secondarily leading to cone death. It has long been assumed that the surviving neural retina is largely resistant to this sensory deafferentation. New evidence from fast retinal degenerations reveals that subtle plasticities in neuronal form and connectivity emerge early in disease. By screening mature natural, transgenic, and knockout retinal degeneration models with computational molecular phenotyping, we have found an extended late phase of negative remodeling that radically changes retinal structure. Three major transformations emerge: 1) Müller cell hypertrophy and elaboration of a distal glial seal between retina and the choroid/retinal pigmented epithelium; 2) apparent neuronal migration along glial surfaces to ectopic sites; and 3) rewiring through evolution of complex neurite fascicles, new synaptic foci in the remnant inner nuclear layer, and new connections throughout the retina. Although some neurons die, survivors express molecular signatures characteristic of normal bipolar, amacrine, and ganglion cells. Remodeling in human and rodent retinas is independent of the initial molecular targets of retinal degenerations, including defects in the retinal pigmented epithelium, rhodopsin, or downstream phototransduction elements. Although remodeling may constrain therapeutic intervals for molecular, cellular, or bionic rescue, it suggests that the neural retina may be more plastic than previously believed. J. Comp. Neurol. 464:1–16, 2003.

Retinal remodeling in inherited photoreceptor degenerations.

Photoreceptor degenerations initiated in rods or the retinal pigmented epithelium usually evoke secondary cone death and sensory deafferentation of the surviving neural retina. In the mature central nervous system, deafferentation evokes atrophy and connective re-patterning. It has been assumed that the neural retina does not remodel, and that it is a passive survivor. Screening of advanced stages of human and rodent retinal degenerations with computational molecular phenotyping has exposed a prolonged period of aggressive negative remodeling in which neurons migrate along aberrant glial columns and seals, restructuring the adult neural retina (1). Many neurons die, but survivors rewire the remnant inner plexiform layer (IPL), forming thousands of novel ectopic microneuromas in the remnant inner nuclear layer (INL). Bipolar and amacrine cells engage in new circuits that are most likely corruptive. Remodeling in human and rodent retinas emerges regardless of the molecular defects that initially trigger retinal degenerations. Although remodeling may constrain therapeutic intervals for molecular, cellular, or bionic rescue, the exposure of intrinsic retinal remodeling by the removal of sensory control in retinal degenerations suggests that neuronal organization in the normal retina may be more plastic than previously believed.

Molecular Phenotyping of Retinal Ganglion Cells

Classifying all of the ganglion cells in the mammalian retina has long been a goal of anatomists, physiologists, and cell biologists. The rabbit retinal ganglion cell layer was phenotyped using intrinsic small molecule signals (aspartate, glutamate, glycine, glutamine, GABA, and taurine) and glutamate receptor-gated 1-amino-4-guanidobutane excitation signals as the clustering dimensions for formal classification. Intrinsic signals alone yielded 7 ganglion cell superclasses and 1 amacrine cell superclass; the addition of excitation signals ultimately resolved 14 natural ganglion cell classes and 3 amacrine cell classes. Ganglion cells comprise two-thirds to three-quarters of the cells in the ganglion cell layer and exhibited distinct metabolic, coupling, and excitation phenotypes, as well as characteristic sizes, population fractions, and patterns. Metabolic signatures (mixtures of glutamate, aspartate, glutamine, and GABA) chemically discriminated ganglion from amacrine cells. Coupling signatures reflected heterologous coupling states across ganglion cells: (1) uncoupled, (2) coupled to GABAergic amacrine cells, and (3) coupled to glycinergic amacrine cells. Excitation signatures reflected differential channel permeation rates across classes after AMPA activation. Extraction of unique size and patterning features from the data sets further validated the robustness of the classification. Because the classifications were explicitly blinded to structure, this is strong evidence that molecular phenotype classes are natural classes. Correspondences of molecular phenotype classes to functional classes were inferred from size, coupling, encounter, and physiological attributes. Ganglion cell classes display markedly different ionotropic drives, which may partly explain the physiological brisk–sluggish spectrum of ganglion cell spiking patterns.

Exploring the retinal connectome

The synthesis and evaluation of 5α-reductase inhibitory activity of some 4-azasteroid-20-ones and 20-oximes and 3β-hydroxy-, 3β-acetoxy-, or epoxy-substituted C21 steroidal 20-ones and 20-oximes having double bonds in the A and/or B ring are described. Inhibitory activity of synthesized compounds was assessed using 5α-reductase enzyme and [1,2,6,7-3H]testosterone as substrate. All synthesized compounds were less active than finasteride (IC50: 1.2 nM). Three 4-azasteroid-2-oximes (compounds 4, 6 and 8) showed good inhibitory activity (IC50: 26, 10 and 11 nM) and were more active than corresponding 4-azasteroid 20-ones (compounds 3, 5 and 7). 3β-Hydroxy-, 3β-acetoxy- and 1α,2α-, 5α,6α- or 6α,7α-epoxysteroid-20-one and -20-oxime derivatives having double bonds in the A and/or B ring showed no inhibition of 5α-reductase enzyme.

A computational framework for ultrastructural mapping of neural circuitry.

Circuitry mapping of metazoan neural systems is difficult because canonical neural regions (regions containing one or more copies of all components) are large, regional borders are uncertain, neuronal diversity is high, and potential network topologies so numerous that only anatomical ground truth can resolve them. Complete mapping of a specific network requires synaptic resolution, canonical region coverage, and robust neuronal classification. Though transmission electron microscopy (TEM) remains the optimal tool for network mapping, the process of building large serial section TEM (ssTEM) image volumes is rendered difficult by the need to precisely mosaic distorted image tiles and register distorted mosaics. Moreover, most molecular neuronal class markers are poorly compatible with optimal TEM imaging. Our objective was to build a complete framework for ultrastructural circuitry mapping. This framework combines strong TEM-compliant small molecule profiling with automated image tile mosaicking, automated slice-to-slice image registration, and gigabyte-scale image browsing for volume annotation. Specifically we show how ultrathin molecular profiling datasets and their resultant classification maps can be embedded into ssTEM datasets and how scripted acquisition tools (SerialEM), mosaicking and registration (ir-tools), and large slice viewers (MosaicBuilder, Viking) can be used to manage terabyte-scale volumes. These methods enable large-scale connectivity analyses of new and legacy data. In well-posed tasks (e.g., complete network mapping in retina), terabyte-scale image volumes that previously would require decades of assembly can now be completed in months. Perhaps more importantly, the fusion of molecular profiling, image acquisition by SerialEM, ir-tools volume assembly, and data viewers/annotators also allow ssTEM to be used as a prospective tool for discovery in nonneural systems and a practical screening methodology for neurogenetics. Finally, this framework provides a mechanism for parallelization of ssTEM imaging, volume assembly, and data analysis across an international user base, enhancing the productivity of a large cohort of electron microscopists.

Generation of an Inbred Miniature Pig Model of Retinitis Pigmentosa

PURPOSE The Pro23His (P23H) rhodopsin (RHO) mutation underlies the most common form of human autosomal dominant retinitis pigmentosa (adRP). The objective of this investigation was to establish a transgenic miniature swine model of RP using the human P23H RHO gene. METHODS Somatic cell nuclear transfer (SCNT) was used to create transgenic miniature pigs that expressed the human P23H RHO mutation. From these experiments, six transgenic founders were identified whose retinal function was studied with full-field electroretinography (ffERG) from 3 months through 2 years. Progeny from one founder were generated and genotyped to determine transgene inheritance pattern. Retinal mRNA was isolated, and the ratio of P23H to wild-type pig RHO was measured. RESULTS A single transgene integration site was observed for five of the six founders. All founders had abnormal scotopic and photopic ffERGs after 3 months. The severity of the ffERG phenotype was grouped into moderately and severely affected groups. Offspring of one founder inherited the transgene as an autosomal dominant mutation. mRNA analyses demonstrated that approximately 80% of total RHO was mutant P23H. CONCLUSIONS Expression of the human RHO P23H transgene in the retina creates a miniature swine model with an inheritance pattern and retinal function that mimics adRP. This large-animal model can serve as a novel tool for the study of the pathogenesis and therapeutic intervention in the most common form of adRP.

Detection of neuron membranes in electron microscopy images using a serial neural network architecture

Study of nervous systems via the connectome, the map of connectivities of all neurons in that system, is a challenging problem in neuroscience. Towards this goal, neurobiologists are acquiring large electron microscopy datasets. However, the shear volume of these datasets renders manual analysis infeasible. Hence, automated image analysis methods are required for reconstructing the connectome from these very large image collections. Segmentation of neurons in these images, an essential step of the reconstruction pipeline, is challenging because of noise, anisotropic shapes and brightness, and the presence of confounding structures. The method described in this paper uses a series of artificial neural networks (ANNs) in a framework combined with a feature vector that is composed of image intensities sampled over a stencil neighborhood. Several ANNs are applied in series allowing each ANN to use the classification context provided by the previous network to improve detection accuracy. We develop the method of serial ANNs and show that the learned context does improve detection over traditional ANNs. We also demonstrate advantages over previous membrane detection methods. The results are a significant step towards an automated system for the reconstruction of the connectome.

Effect of shape and coating of a subretinal prosthesis on its integration with the retina

Retinal stimulation with high spatial resolution requires close proximity of electrodes to target cells. This study examines the effects of material coatings and 3-dimensional geometries of subretinal prostheses on their integration with the retina. A trans-scleral implantation technique was developed to place microfabricated structures in the subretinal space of RCS rats. The effect of three coatings (silicon oxide, iridium oxide and parylene) and three geometries (flat, pillars and chambers) on the retinal integration was compared using passive implants. Retinal morphology was evaluated histologically 6 weeks after implantation. For 3-dimensional implants the retinal cell phenotype was also evaluated using Computational Molecular Phenotyping. Flat implants coated with parylene and iridium oxide were generally well tolerated in the subretinal space, inducing only a mild gliotic response. However, silicon-oxide coatings induced the formation of a significant fibrotic seal around the implants. Glial proliferation was observed at the base of the pillar electrode arrays and inside the chambers. The non-traumatic penetration of pillar tips into the retina provided uniform and stable proximity to the inner nuclear layer. Retinal cells migrated into chambers with apertures larger than 10 mum. Both pillars and chambers achieved better proximity to the inner retinal cells than flat implants. However, isolation of retinal cells inside the chamber arrays is likely to affect their long-term viability. Pillars demonstrated minimal alteration of the inner retinal architecture, and thus appear to be the most promising approach for maintaining close proximity between the retinal prosthetic electrodes and target neurons.

Retinal remodeling in human retinitis pigmentosa.

Retinitis Pigmentosa (RP) in the human is a progressive, currently irreversible neural degenerative disease usually caused by gene defects that disrupt the function or architecture of the photoreceptors. While RP can initially be a disease of photoreceptors, there is increasing evidence that the inner retina becomes progressively disorganized as the outer retina degenerates. These alterations have been extensively described in animal models, but remodeling in humans has not been as well characterized. This study, using computational molecular phenotyping (CMP) seeks to advance our understanding of the retinal remodeling process in humans. We describe cone mediated preservation of overall topology, retinal reprogramming in the earliest stages of the disease in retinal bipolar cells, and alterations in both small molecule and protein signatures of neurons and glia. Furthermore, while Müller glia appear to be some of the last cells left in the degenerate retina, they are also one of the first cell classes in the neural retina to respond to stress which may reveal mechanisms related to remodeling and cell death in other retinal cell classes. Also fundamentally important is the finding that retinal network topologies are altered. Our results suggest interventions that presume substantial preservation of the neural retina will likely fail in late stages of the disease. Even early intervention offers no guarantee that the interventions will be immune to progressive remodeling. Fundamental work in the biology and mechanisms of disease progression are needed to support vision rescue strategies.