Biology subjects

Pugh, E. N.

Publications and source records attributed to Pugh, E. N..

5 recordsLinked to original sources

bioRxiv2026

Optoretinography in R9AP-bradyopsia reveals the essential role of G-protein signaling in the human cone elongation response

Human cone and rod outer segments exhibit a rapid shrinkage followed by a slower elongation in response to light, forming the basis of the optoretinogram. The molecular basis of this optical assay of photoreceptor function remains incompletely understood. In mouse rods, the elongation response requires transducin, the G-protein -subunit activated in the initial amplifying step of the phototransduction cascade. Here, we measured human cone outer segment responses in subjects with bradyopsia arising from a triple-deletion mutation in R9AP, the anchor and transport protein for the GTPase-activating protein (GAP) RGS9. Immunoprecipitation showed that the mutant R9AP has greatly reduced affinity for RGS9, predictably reducing the level of RGS9 in the outer segment. The bradyopsia subjects elongation responses had normal activation kinetics, amplitude and photosensitivity, but markedly slowed recovery. These results indicate that normal levels of RGS9 are required for deactivation of the cone elongation response, situating the underlying molecular mechanism within the G-protein cascade at or prior to formation of the GAP complex. The recovery of the cone elongation response, measured in a paired-flash paradigm for stimuli isomerizing up to 75% of opsin, revealed that elongation requires a substrate that is depleted and recovers in a bleach-dependent manner. In particular, recovery from the highest bleaching exposure (75%) tracked the time course of cone opsin regeneration, implying that unregenerated cone opsin produces "dark light", known in rods to arise from constitutive activation of G-protein. Taken together, the results from controls and bradyopsia identify the inactive (GDP-bound) transducin complex as the essential substrate for the human cone elongation response. Suppression of the elongation response in the paired-flash paradigm further revealed the complete profile of the initial fast cone outer segment shrinkage response and reinforced its origin in opsin structural changes. Overall, these results identify key mechanistic elements of the optoretinogram and enable its use as a molecularly interpretable, non- invasive assay of cone function in disease and therapeutic response.

neuroscience↗

bioRxiv2025

Biophysical Basis of the in vivo ERG of the Mouse: Rod, RPE and Müller glial Cell Contributions To Dark and to Light-driven Changes in Sub-Retinal Space o

The subretinal space (SRS) is a contiguous extracellular volume bounded by the external limiting membrane (ELM) and the retinal pigment epithelium (RPE). Approximately 90% of mouse rod photoreceptor dark current circulates in the SRS, and rod photoresponses materially alter SRS concentrations of permeant ions, especially K+. Following intravitreal injection of drugs that block glutamatergic synapses, and drugs that inhibit Kir4.1 and Kir7.1 channels expressed in Muller and RPE cells respectively, we measured electroretinograms (ERGs) in response to light flashes that completely suppress rod dark current, and compared ERG c-waves with predictions of a biophysical model of extracellular current sources generated by rod, Muller and RPE cells. SRS K+ fluxes in the dark steady-state were dominated by rods - NKX (-6.6 mM s-1), Kv2.1 channels (+5.4 mM s-1), NCKX (0.3 mM s-1) and HCN1 channel fluxes (1.0 mM s-1) - with much smaller contributions from Kir4.1 and Kir7.1 channels and trans-ELM and trans-RPE fluxes combining to zero net flux at resting SRS Ko of 5 mM (re 3.5 mM [K+] in blood plasma). In response to rod-saturating stimulation, Ko was predicted to decline to [~]3.5 mM with a half-time of [~] 0.5 s, driven mainly by a hyperpolarization-dependent decline in Kv2.1 current. The kinetics and amplitude of the c-wave are explained by the decline in SRS Ko, which negatively shifts the Nernst potential of apical RPE Kir7.1 channels, increasing outward current that sinks through the paracellular resistance to basal Bestrophin and CFTR chloride channels causing a trans-epithelial potential of [~] 2 mV.

biophysics↗

bioRxiv2025

Biophysical Basis of the in vivo Electroretinogram of the Mouse: Current Source Density Analysis of Genetically and Pharmacologically Isolated Rod photoreceptor-driven Currents

To better understand the molecular basis of the mouse electroretinogram (ERG) we have developed a biophysical model of the rod photoreceptor layers ionic mechanisms and applied current source-density (CSD) analysis to predict the genetically and pharmacologically isolated rod ERG a-wave. The saturating a-wave is characterized by a rapid relaxation ({tau} ~ 6 ms) from its maximum that has been hypothesized to be caused by HCN1 channel opening consequent to light-triggered sustained hyperpolarization, or by extracellular flow of capacitive current during hyperpolarization. To test these hypotheses, the CSD model included an ensemble of 11 rods with cell body locations spanning the outer nuclear layer, and with ionic mechanisms -- including CNG channels, NCKX in the outer segment, NKX, Kv2.1 channels in the inner segment, and HCN1 throughout the "non-outer segment" -- fully specified as to axial distributions, voltage dependencies, and the extracellular conductivity of the photoreceptor layer extracellular space. Predicting with CSD the steady-state axial distributions of rod dark current and transretinal potential, and the spatio-temporal response of the activation phase of the rods light response to intense stimuli, the analysis confirmed the Robson-Frishman hypothesis that extracellular capacitive current flowing towards the inner segment upon the rapid closure of CNG current plays a major role in shaping the early a-wave. The CSD analysis also reveals that extracellular current from HCN1 channels opened by the hyperpolarization contributes a net extracellular current flowing toward the ONL that contributes materially to the saturated a-wave relaxation and to setting its plateau level.

biophysics↗

bioRxiv2025

Biophysical Basis of the in vivo Electroretinogram of the Mouse: 4-Shell Current Source Density Model

A goal of contemporary physiology is to translate the knowledge obtained ex vivo of the molecular structure and function of ionic mechanisms into tools for quantitative, in vivo measurement of that function in humans, and in animal models of disease and therapeutic intervention. Non-invasive field potentials such as ECGs, EMGs, EEGs, and ERGs hold great promise in efforts to achieve this goal, but their full translation is challenging due to the multiplicity of cells with distinct ionic mechanisms and distributions of membrane current sources and sinks, and the requirement of adequate characterization of volume conduction in the relevant tissue(s). The molecular identities and subcellular distributions of the ionic mechanisms of mouse rod photoreceptors and the adjacent retinal pigment epithelium (RPE) have been thoroughly characterized, and are associated with two major components of the ERG, the a-wave and the c-wave, respectively. To develop a molecular-biophysical description of these components of the ERG, we have pharmacologically and genetically isolated rod photoreceptor-driven currents, created a 3D "4-shell" model of volume conduction in the mouse eye and extraocular tissue, and solved the Equation of Continuity for trans-photoreceptor layer and trans-RPE layer sources. Corneal and intraocular measurements of a- and c-waves are shown to reject the classic Rodieck-Ford electrical circuit model of the ERG, but found consistent with a 4-shell model having realistic values for extracellular conductivity in the eye and extraocular tissues. Our results and analysis explain the large variation across studies in maximal a-wave amplitudes and show how a-wave amplitudes exceeding 1 mV can be achieved.

biophysics↗

bioRxiv2018

Aperture Phase Modulation with Adaptive Optics: A Novel Approach for Speckle Reduction and Structure Extraction in Optical Coherence Tomography

AbstractSpeckle is an inevitable consequence of the use of coherent light in optical coherence tomography (OCT), and often acts as noise that obscures micro-structures of biological tissue. We here present a novel method of suppressing speckle noise intrinsically compatible with adaptive optics (AO) in OCT system: by modulating the phase inside the imaging system pupil aperture with a segmented deformable mirror, thus producing minor perturbations in the point spread function (PSF) to create un-correlated speckle pattern between B-scans, and further averaging to wash out the speckle but maintain the structures. It is a well-controlled and universal method which can efficiently determine the optimal range of phase modulation that minimizing speckle noise while maximizing image resolution and signal strength for different systems and/or samples. As an active method, its effectiveness and efficiency were demonstrated by both ex-vivo non-biological and in-vivo biological applications.

bioengineering↗