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Robert E Marc

Publications and source records attributed to Robert E Marc.

10 recordsLinked to original sources

Excitation mapping with the organic cation AGB2+.

Excitation mapping is a method of visualizing the signaling history of neurons with permeant organic cations. It is compatible with high-resolution imaging, allowing concurrent visualization of all neuronal classes and their glutamate-gated excitation histories. Excitation mapping documents the stability and precision of neuronal signaling within a given neuronal class, arguing that single unit electrophysiological sampling accurately reflects neuronal diversity. We here review the theory of excitation mapping, provide methods and protocol links; outline imaging concepts; provide parametric data on the temporal range and physiological sensitivity of excitation mapping; and show that immunocytochemical methods for macromolecules are compatible with excitation mapping.

Agmatine↗

Retinal remodeling during retinal degeneration.

Retinal degenerations, regardless of the initiating event or gene defect, often result in a loss of photoreceptors. This formal deafferentation of the neural retina eliminates the intrinsic glutamatergic drive of the sensory retina and, perhaps more importantly, removes coordinated Ca++-coupled signaling to the neural retina. As in other central nervous system degenerations, deafferentation activates remodeling. Neuronal remodeling is the common fate of all photoreceptor degenerations.

Animals↗

Retinal remodelling.

Retinal degenerative diseases that progress through loss of photoreceptors initiate a sequence of events that culminates in negative remodelling of the retina. Initially, photoreceptor loss ablates glutamatergic signalling to the neural retina and eliminates coordinate Ca++-coupled homeostatic signalling. Retinal neurons react to this loss of glutamatergic input through retinal rewiring and migration of neurons throughout the axis of the retina. All diseases that kill photoreceptors trigger retinal remodelling as the final common pathway and cell death is a common feature. Retinal remodelling resembles CNS pathologic remodelling and constitutes a major challenge to all rescue strategies.

Amacrine Cells↗

Contribution of a glial glutamate transporter to GABA synthesis in the retina.

Neuronal glutamate transporters have been shown to play a role in GABA synthesis by enhancing glutamate uptake. In the present study, we have examined whether a glial glutamate transporter, GLAST, has a role in GABA synthesis in the mammalian retina. We found that the retinal GABA level was about two-fold higher in the GLAST-/- mouse retina compared to that in the wild type. Endogenous glutamate level was also increased about 2-fold in the mutant. Therefore, loss of GLAST results in a higher retinal GABA level, probably due to increased availability of its precursor, glutamate. An increase in GABAergic activity can be expected to affect trigger features such as directional selective response of neurons in the GLAST-/- mouse retina.

Amino Acid Transport System X-AG↗

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.

Aging↗

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.

Animals↗

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.

Amacrine Cells↗

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.

Agmatine↗

Gamma-glutamyl transferase deficiency results in lung oxidant stress in normoxia.

gamma-Glutamyl transferase (GGT) is critical to glutathione homeostasis by providing substrates for glutathione synthesis. We hypothesized that loss of GGT would cause oxidant stress in the lung. We compared the lungs of GGT(enu1) mice, a genetic model of GGT deficiency, with normal mice in normoxia to study this hypothesis. We found GGT promoter 3 (P3) alone expressed in normal lung but GGT P3 plus P1, an oxidant-inducible GGT promoter, in GGT(enu1) lung. Glutathione content was barely decreased in GGT(enu1) lung homogenate and elevated nearly twofold in epithelial lining fluid, but the fraction of oxidized glutathione was increased three- and fourfold, respectively. Glutathione content in GGT(enu1) alveolar macrophages was decreased nearly sixfold, and the oxidized glutathione fraction was increased sevenfold. Immunohistochemical studies showed glutathione deficiency together with an intense signal for 3-nitrotyrosine in nonciliated bronchiolar epithelial (Clara) cells and expression of heme oxygenase-1 in the vasculature only in GGT(enu1) lung. When GGT(enu1) mice were exposed to hyperoxia, survival was decreased by 25% from control because of accelerated formation of vascular pulmonary edema, widespread oxidant stress in the epithelium, diffuse depletion of glutathione, and severe bronchiolar cellular injury. These data indicate a critical role for GGT in lung glutathione homeostasis and antioxidant defense in normoxia and hyperoxia.

Animals↗

Functionally intact glutamate-mediated signaling in bipolar cells of the TRKB knockout mouse retina.

In the juvenile trkB knockout (trkB-/-) mouse, retina synaptic communication from rods to bipolar cells is severely compromised as evidenced by a complete absence of electroretinogram (ERG) b-wave, even though the inner retina appears anatomically normal (Rohrer et al., 1999). Since it is well known that the b-wave reflects light-dependent synaptic activation of ON bipolar cells via their metabotropic glutamate receptor, mGluR6, we sought to analyze the anatomical and functional integrity of the glutamatergic synapses at these and other bipolar cells in the trkB(-/-) mouse. Although rod bipolar cells from wild-type juvenile mice were determined to be immunopositive for trkB, postsynaptic metabotropic and ionotropic glutamate receptor-mediated pathways in ON and OFF bipolar cells were found to be functionally intact, based on patch electrode recordings, using brief applications ("puffs") of glutamate or its analog, 2-amino-4-phosphonobutyric acid (APB), a selective agonist for mGluR6 receptors. Ionotropic glutamate receptor function was assayed in OFF-cone bipolar and horizontal cells by applying exogenous glutamatergic agonists in the presence of the channel-permeant guanidinium analogue, 1-amino-4-guanidobutane (AGB). Electron-microscopic analysis revealed that the ribbon synapses between rods and postsynaptic rod bipolar and horizontal cells were formed at the appropriate age and appear to be structurally intact, and immunohistochemical analysis did not detect profound defects in the expression of excitatory amino acid transporters involved in glutamate clearance from the synaptic cleft. These data indicate that there does not appear to be evidence for postsynaptic deficits in glutamatergic signaling in the ON and OFF bipolar cells of mice lacking trkB.

Agmatine↗