Biomedical subjects

David R Pepperberg

Publications and source records attributed to David R Pepperberg.

8 recordsLinked to original sources

PubMed2004

Neurotransmitter analog tethered to a silicon platform for neuro-BioMEMS applications.

The design of chemically well-defined, machinable surfaces containing neuroactive molecules offers potential for fundamental neuroscience and clinical neural engineering applications. Here we report the assembly and characterization of silicon platforms containing a tethered form of muscimol. Muscimol, an analog of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA), is a potent agonist at postsynaptic GABA(A) and GABA(C) receptors. Surfaces were assembled using covalent avidin conjugation to silanized silicon followed by high-affinity avidin-biotin binding of a biotinylated derivative of muscimol (muscimol-biotin). Contact angle measurements, ellipsometry, and X-ray photoelectron spectroscopy (XPS) were conducted to characterize the wettability, thickness, and chemical composition of progressively deposited surface layers. The data demonstrate successful incorporation of a neurotransmitter analog as part of a layered, silicon-based structure possessing robust and specific biomolecular composition. These findings represent a step toward the design of platforms for applications involving control and modulation of neural signaling.

Avidin↗

PubMed2004

Step response of mouse rod photoreceptors modeled in terms of elemental photic signals.

The process of light adaptation in rod photoreceptors enables these sensory cells of the retina to remain responsive to photic stimuli over a broad range of light intensity. Recent studies have employed the technique of paired-flash electroretinography to determine properties of phototransduction, and of light and dark adaptation, in rod photoreceptors in the living eye. Building on these studies, we have developed a theoretical model aimed at explaining the rod electrical response to a step of light based on known physiology. The central feature of the model is its description of the macroscopic (i.e., measured) response in terms of a time-evolving, weighted sum of elemental responses determined under dark-adapted and near fully light-adapted conditions. The model yields a time-dependent function that describes the course of desensitization and putatively represents the cumulative dynamics of underlying biochemical processes involved in light adaptation of the rod.

Action Potentials↗

PubMed2004

Dark adaptation of rod photoreceptors in normal subjects, and in patients with Stargardt disease and an ABCA4 mutation.

PURPOSE: Psychophysical and electroretinographic (ERG) studies indicate that patients with Stargardt disease exhibit abnormally slow rod dark adaptation after illumination that bleaches a substantial fraction of rhodopsin. However, relatively little information is available concerning rod recovery in this disease after weaker adapting (i.e., conditioning) light. With the use of a paired-flash ERG method, properties of the derived rod response to a low-bleach (<1%) but rod-saturating conditioning flash were investigated in seven normal subjects and in five Stargardt patients with identified sequence variations in the ABCA4 gene. METHODS: In the first of two experiments, the interval between a fixed conditioning flash (67 or 670 scotopic cd s m(-2)) and a bright probe flash of fixed strength was varied to determine the falling-phase kinetics of the derived rod response to the conditioning flash. In the second, the instantaneous amplitude-intensity function for the rod response at an intermediate stage of recovery from the conditioning flash was determined by presenting a test flash of various strengths at a fixed time after the conditioning flash, and a probe flash at 200 ms after the test flash. RESULTS: The maximum peak amplitude of the dark-adapted, rod-mediated a-wave determined in Stargardt patients (211 +/- 87 microV) was on average lower than that determined in normal subjects (325 +/- 91 microV; P = 0.06). The derived rod response to the 670 scotopic cd s m(-2) conditioning flash determined in normal subjects and Stargardt patients exhibited a biphasic recovery, and the kinetics of the early stage of this recovery were similar in the two subject groups. For both normal subjects and patients, normalized amplitude-intensity functions describing the dark-adapted derived rod response exhibited half-saturation at approximately 1.5 log scotopic troland second. In both groups, the normalized amplitude-intensity function determined at approximately 2 seconds after the 67 scotopic cd s m(-2) conditioning flash and at approximately 9 seconds after the 670 scotopic cd s m(-2) conditioning flash exhibited an average desensitization (i.e., an increase of test flash strength at half-saturation) of approximately 0.5 to 0.6 log unit relative to that determined under dark-adapted conditions. CONCLUSIONS: The results indicate that, despite a reduction in the average dark-adapted maximum a-wave amplitude in the Stargardt/ABCA4 patients, the early-stage recovery kinetics of the derived rod response to a low-bleaching conditioning flash as well as the lingering rod desensitization produced by such a flash are similar to those determined in normal subjects.

ATP-Binding Cassette Transporters↗

PubMed2003

Assembly and characterization of biofunctional neurotransmitter-immobilized surfaces for interaction with postsynaptic membrane receptors.

Herein, we report progress toward the development of bioactive surfaces based on gamma-aminobutyric acid (GABA), a major neurotransmitter in the nervous system. Whereas immobilization techniques have focused largely on antibodies, enzymes, and receptors, to our knowledge, this is the first report of a prototype neurotransmitter-immobilized surface. Biosurfaces were assembled onto either mica or glass using passive adsorption of avidin and subsequent attachment of a derivatized form of GABA via a biotin-avidin affinity bond. Surface characterization of these prepared bimolecular surfaces was determined using atomic force microscopy in tapping mode. The data reveal that passive adsorption of avidin is uniformly dispersed and cluster densities can be controlled through the concentration of the avidin incubation solution. GABA tethered via biotin to these avidin surfaces displayed a unique surface topology; in addition, histograms of surface heights suggest two different types of molecular cluster populations. Functional assays were performed to test the biological activity of the synthesized GABA. Anti-GABA antibody directed to these bimolecular surfaces result in morphological topologies and histograms that indicate antibody-antigen binding. However, nonspecific anti-immunoglobulin G antibodies directed to these surfaces show low binding affinity. Taken together, the data support the idea that the synthesized surfaces are biofunctional.

Adsorption↗

PubMed2003

Bleaching desensitization: background and current challenges.

"Bleaching desensitization" in rod photoreceptors refers to the prolonged depression of phototransduction sensitivity exhibited by rods after their exposure to bright light, i.e., after photolysis (bleaching) of a substantial fraction of rhodopsin in the outer segments. Rod recovery from bleaching desensitization depends critically on operation of the retinoid visual cycle: in particular, on the removal of all-trans retinal bleaching product from opsin and on the delivery of 11-cis retinal to opsin's chromophore binding site. The present paper summarizes representative findings that address the mechanism of bleaching desensitization.

Dark Adaptation↗

PubMed2003

Acute radiolabeling of retinoids in eye tissues of normal and rpe65-deficient mice.

PURPOSE: Mice with a targeted disruption of the gene encoding RPE65, a protein ordinarily highly expressed in the retinal pigment epithelium (RPE), accumulate abnormally high levels of all-trans retinyl ester in the RPE and exhibit very little 11-cis retinal in the retina. The present study was undertaken to determine whether the Rpe65-deficient mouse exhibits an abnormal flux of retinoid between the systemic circulation and the eye tissues. METHODS: Dark-adapted Rpe65-deficient mice (Rpe65(-/-)) and wild-type control mice (Rpe65(+/+)) of approximate ages 1 and 3 months received an intraperitoneal injection of all-trans ((3)H)retinol. The mice were maintained in darkness for a defined period ( approximately 1.5, 4.5, 24, or 48 hours) and then anesthetized, exsanguinated, and killed. Retinoids contained in the retina, RPE, serum, and liver were extracted and analyzed for ((3)H) radioactivity and molar level. RESULTS: The specific activity (SA, in counts per minute per nanomole) of serum all-trans ((3)H)retinol in all mice exhibited a peak at postinjection times of 1.5 or 4.5 hours, and by 48 hours declined to approximately 7% or less of the peak. In Rpe65(+/+) mice, the average SA of RPE ((3)H)retinyl ester similarly exhibited an early peak (4.5 hours) and by 48 hours declined to approximately 6% to 10% of the peak. By contrast, the average SA of RPE ((3)H)retinyl ester in Rpe65(-/-) mice exhibited a peak at 24 or 48 hours. Radioactivity and molar data for serum all-trans retinol and RPE retinyl ester obtained at 4.5 hours were analyzed to infer the molar influx of all-trans retinol from the circulation into the RPE. Levels of all-trans retinol influx derived from this analysis (mean +/- SD: 0.014 +/- 0.004 nmol in 1-month Rpe65(+/+) mice; 0.021 +/- 0.009 nmol in 1-month Rpe65(-/-) mice; 0.016 +/- 0.013 nmol in 3-month Rpe65(+/+) mice; 0.026 +/- 0.018 nmol in 3-month Rpe65(-/-) mice) did not differ significantly from one another (P > 0.169). However, the inferred fractional influx (molar amount of entering all-trans retinol divided by the molar amount of RPE retinyl ester) in Rpe65(+/+) animals (0.34 +/- 0.04 and 0.10 +/- 0.03, respectively, for 1- and 3-month mice) substantially exceeded that for Rpe65(-/-) animals (0.055 +/- 0.023 and 0.015 +/- 0.006, respectively, for 1- and 3-month mice). Significant levels of ((3)H)retinaldehydes were detected in the retinas of Rpe65(+/+) mice, but not in those of Rpe65(-/-) mice, after the longer postinjection periods. CONCLUSIONS: The results indicate preservation of a substantial inward flux of all-trans retinol from the circulation into the RPE of Rpe65(-/-) mice, despite the presence of abnormally high molar levels of RPE retinyl ester. They further imply the occurrence of a robust outward movement of all-trans retinol from the RPE into the circulation in Rpe65(+/+) mice, and substantial impairment of this efflux process in Rpe65(-/-) mice. These findings raise the hypothesis that in normal RPE, 11-cis retinal and/or 11-cis retinol stimulate the efflux of all-trans retinol at the RPE basolateral membrane. In 3-month Rpe65(+/+) mice, the observed relationship between the SAs of retinaldehydes in the retina and of RPE retinyl ester is consistent with a last-in/first-out processing of all-trans retinol to 11-cis retinal within normally functioning RPE.

Animals↗

PubMed2002

Excitation and desensitization of mouse rod photoreceptors in vivo following bright adapting light.

Electroretinographic (ERG) methods were used to determine response properties of mouse rod photoreceptors in vivo following adapting illumination that produced a significant extent of rhodopsin bleaching. Bleaching levels prevailing at approximately 10 min and approximately 20 min after the adapting exposure were on average 14 % and 9 %, respectively, based on the analysis of visual cycle retinoids in the eye tissues. Recovery of the rod response to the adapting light was monitored by analysing the ERG a-wave response to a bright probe flash presented at varying times during dark adaptation. A paired-flash procedure, in which the probe flash was presented at defined times after a weak test flash of fixed strength, was used to determine sensitivity of the rod response to the test flash. Recovery of the response to the adapting light was 80 % complete at 13.5 +/- 3.0 min (mean +/- S.D.; n = 7) after adapting light offset. The adapting light caused prolonged desensitization of the weak-flash response derived from paired-flash data. By comparison with results obtained in the absence of the adapting exposure, desensitization determined with a test-probe interval of 80 ms was ~fourfold after 5 min of dark adaptation and approximately twofold after 20 min. The results indicate, for mouse rods in vivo, that the time scale for recovery of weak-flash sensitivity substantially exceeds that for the recovery of circulating current following significant rhodopsin bleaching. The lingering desensitization may reflect a reduced efficiency of signal transmission in the phototransduction cascade distinct from that due to residual excitation.

Adaptation, Physiological↗

PubMedDate not supplied

Conditioning light differentially desensitizes rod phototransduction mediated by native and 9-demethyl analog visual pigment.

Light adaptation in rod photoreceptors is thought to involve down-regulation of the signaling activity of photoactivated rhodopsin (R*). However, electrophysiological evidence in support of this notion has come largely from studies of truncated, perfused rod outer segments and of rods genetically engineered to perturb known steps in R* deactivation. To test this hypothesis within intact native rods, we examined the effect of a fixed conditioning flash on rods prepared to contain 9-demethyl rhodopsin (9dR) in addition to residual rhodopsin. 9dR, an opsin-based photopigment containing 11-cis 9-demethylretinal as its chromophore, exhibits a blue-shifted excitation spectrum and sluggish deactivation kinetics, properties that distinguish the signaling activities of photoactivated 9dR (9dR*) from those of R*. Saturating photocurrent responses mediated preferentially by R* and 9dR* were obtained with test flash stimulation at 640 and 440 nm, respectively, under dark-adapted conditions (unconditioned response) and at a fixed time after a 640-nm conditioning flash of fixed high intensity. At each test wavelength, the decrease in photocurrent saturation period induced by the conditioning flash was analyzed to determine psi, the sensitivity of the conditioned response relative that of the unconditioned response; psi640/psi440, the ratio of relative sensitivities, was then obtained. Data obtained from 12 rods yielded psi640/psi440 = 0.60 +/- 0.13 (mean +/- SD). As common pools of transducin and other downstream components mediate transduction initiated by both R* and 9dR*, the finding that psi640 < psi440 provides direct evidence for the down-regulation specifically of R*'s signaling activity during rod light adaptation.

Adaptation, Ocular↗