Characterization of pupil responses to blue and red light stimuli in autosomal dominant retinitis pigmentosa due to NR2E3 mutation.

PURPOSE: We characterized the pupil responses that reflect rod, cone, and melanopsin function in a genetically homogeneous cohort of patients with autosomal dominant retinitis pigmentosa (adRP). METHODS: Nine patients with Gly56Arg mutation of the NR2E3 gene and 12 control subjects were studied. Pupil and subjective visual responses to red and blue light flashes over a 7 log-unit range of intensities were recorded under dark and light adaptation. The pupil responses were plotted against stimulus intensity to obtain red-light and blue-light response curves. RESULTS: In the dark-adapted blue-light stimulus condition, patients showed significantly higher threshold intensities for visual perception and for a pupil response compared to controls (P = 0.02 and P = 0.006, respectively). The rod-dependent, blue-light pupil responses decreased with disease progression. In contrast, the cone-dependent pupil responses (light-adapted red-light stimulus condition) did not differ between patients and controls. The difference in the retinal sensitivity to blue and red stimuli was the most sensitive parameter to detect photoreceptor dysfunction. Unexpectedly, the melanopsin-mediated pupil response was decreased in patients (P = 0.02). CONCLUSIONS: Pupil responses of patients with NR2E3-associated adRP demonstrated reduced retinal sensitivity to dim blue light under dark adaptation, presumably reflecting decreased rod function. Rod-dependent pupil responses were quantifiable in all patients, including those with non-recordable scotopic electroretinogram, and correlated with the extent of clinical disease. Thus, the chromatic pupil light reflex can be used to monitor photoreceptor degeneration over a larger range of disease progression compared to standard electrophysiology.

Test-retest repeatability of the pupil light response to blue and red light stimuli in normal human eyes using a novel pupillometer.

In this study, we evaluated the repeatability of pupil responses to colored light stimuli in healthy subjects using a prototype chromatic pupillometer. One eye of 10 healthy subjects was tested twice in the same day using monochromatic light exposure at two selected wavelengths (660 and 470 nm, intensity 300 cd/m(2)) presented continuously for 20 s. Pupil responses were recorded in real-time before, during, and after light exposure. Maximal contraction amplitude and sustained contraction amplitude were calculated. In addition, we quantified the summed pupil response during continuous light stimulation as the total area between a reference line representing baseline pupil size and the line representing actual pupil size over 20 s (area under the curve). There was no significant difference in the repeated measure compared to the first test for any of the pupil response parameters. In conclusion, we have developed a novel prototype of color pupillometer which demonstrates good repeatability in evoking and recording the pupillary response to a bright blue and red light stimulus.

Light responsive multilayer surfaces with controlled spatial extinction capability

Multilayer systems obtained using the Layer-by-Layer (LbL) technology have been proposed for a variety of biomedical applications in tissue engineering and regenerative medicine. LbL assembly is a simple and highly versatile method to modify surfaces and fabricate robust and highly-ordered nanostructured coatings over almost any type of substrates and with a wide range of substances. The incorporation of polyoxometalate (POM) inorganic salts as constituents of the layers presents a possibility of promoting light-stimuli responses in LbL substrates. We propose the design of a biocompatible photo-responsive multilayer system based on a Preyssler-type POM ([NaP5W30O110]14â ) and a natural origin polymer, chitosan, using the LbL methodology. The photo-reduction properties of the POM allow the spatially controlled disruption of the assembled layers due to the weakening of the electrostatic interactions between the layers. This system has found applicability in detaching devices, such as the cell sheet technology, which may solve the drawbacks actually found in other cell treatment proposals.

Determination of Rod and Cone Influence to the Early and Late Dynamic of the Pupillary Light Response.

PURPOSE: This study aims to identify which aspects of the pupil light reflex are most influenced by rods and cones independently by analyzing pupil recordings from different mouse models of photoreceptor deficiency. METHODS: One-month-old wild type (WT), rodless (Rho-/-), coneless (Cnga3-/-), or photoreceptor less (Cnga3-/-; Rho-/- or Gnat1-/-) mice were subjected to brief red and blue light stimuli of increasing intensity. To describe the initial dynamic response to light, the maximal pupillary constriction amplitudes and the derivative curve of the first 3 seconds were determined. To estimate the postillumination phase, the constriction amplitude at 9.5 seconds after light termination was related to the maximal constriction amplitude. RESULTS: Rho-/- mice showed decreased constriction amplitude but more prolonged pupilloconstriction to all blue and red light stimuli compared to wild type mice. Cnga3-/- mice had constriction amplitudes similar to WT however following maximal constriction, the early and rapid dilation to low intensity blue light was decreased. To high intensity blue light, the Cnga3-/- mice demonstrated marked prolongation of the pupillary constriction. Cnga3-/-; Rho-/- mice had no pupil response to red light of low and medium intensity. CONCLUSIONS: From specific gene defective mouse models which selectively voided the rod or cone function, we determined that mouse rod photoreceptors are highly contributing to the pupil response to blue light stimuli but also to low and medium red stimuli. We also observed that cone cells mainly drive the partial rapid dilation of the initial response to low blue light stimuli. Thus photoreceptor dysfunction can be derived from chromatic pupillometry in mouse models.

Nitric oxide photorelease from hydrogels and from skin containing a nitro-ruthenium complex

Nitric oxide (NO) is a gaseous molecule that has specific functions dictated by its localization and its kinetics of release. As NO-donors have a range of potential uses in the skin, much attention has been paid to the development of topical NO delivery systems. The aim of this work was to study the release rate and the skin penetration of the NO-donor cis[Ru(NO(2))(bpy)(2)(4-pic)](+) from different gel formulations and their potential as topical NO delivery systems under light stimuli. Among the formulations developed, the anionic gel retarded the nitro-ruthenium complex diffusion and also obstructed NO release after light irradiation. On the other hand, NO release before light irradiation was observed when the complex was dispersed in the cationic chitosan gel, possibly due to oxi-redox reactions between the amino groups of the polymer and the drug molecule. Finally, the non-ionic gel released the NO after light irradiation to the same extent as a drug aqueous solution at the same pH. The drug dispersed in this gel also penetrated into the stratum corneum skin layer, and the nitro-ruthenium complex present in the skin was able to release the NO after light stimuli, suggesting the potential use of this formulation as a topical NO delivery system. (C) 2010 Elsevier B.V. All rights reserved.

The effect of repeated prepulse and reflex stimulus presentations on startle prepulse inhibition

The magnitude of a startle reflex is inhibited if the reflex-eliciting stimuli is preceded by a prepulse stimulus at a short lead interval. Previous research in humans has shown that the extent of prepulse inhibition decreases over repeated presentations of reflex stimuli and prepulse-reflex stimulus pairings. The present study (N=70) investigated the effect of repeated presentations of prepulse stimuli, reflex stimuli, or prepulse-reflex stimulus pairings on prepulse inhibition. Five groups of subjects were presented during habituation training with either (a) reflex stimuli, (b) prepulse-reflex stimulus pairings, (c) a random sequence of prepulse and reflex stimuli, (d) prepulse stimuli, or (e) experimentally irrelevant light stimuli. Prepulse inhibition was reduced if startle stimuli were presented during habituation ((a), (b), (c)), but not after repeated presentation of the prepulse or the light stimulus ((d), (e)). The reduction in prepulse inhibition was abolished after dishabituation of the startle reflex. The present results indicate that habituation of the startle reflex can result in a reduction of prepulse inhibition. (C) 1998 Elsevier Science B.V.

Serial conditional discrimination and temporal bisection in rats selectively lesioned in the dentate gyrus

In positive serial conditional discrimination, animals respond during a target stimulus when it is preceded by a feature stimulus, but they do not respond when the same target stimulus is presented alone. Moreover, the feature and target stimuli are separated from each other by an empty interval. The present work aimed to investigate if two durations (4 or 16s) of the same feature stimulus (light) could modulate the operant responses of rats to different levers (A and B) during a 5-s target stimulus (tone). In the present study, lever A was associated with the 4-s light, and lever B was associated with the 16-s light. A 5-s empty interval was included between the light and the tone. In the same training procedure, the rats were also presented with the 5-s tone without the preceding light stimuli. In these trials, the responses were not reinforced. We evaluated the hippocampal involvement of these behavioral processes by selectively lesioning the dentate gyrus with colchicine. Once trained, the rats were submitted to a test using probe trials without reinforcement. They were presented with intermediate durations of the feature stimulus (light) to obtain a temporal bisection curve recorded during the exposure to the target stimuli. The rats from both groups learned to respond with high rates during tones preceded by light and with low rates during tones presented alone, which indicated acquisition of the serial conditional discrimination. The rats were able to discriminate between the 4- and 16-s lights by correctly choosing lever A or B. In the test, the temporal bisection curves from both experimental groups showed a bisection point at the arithmetic mean between 4 and 16s. Such processes were not impaired by the dentate gyrus lesion. Thus, our results showed that different durations of a feature stimulus could result in conditional properties. However, this processing did not appear to depend on the dentate gyrus alone. (C) 2011 Published by Elsevier B.V.

Intrinsically photosensitive retinal ganglion cells.

The recent discovery of melanopsin-expressing retinal ganglion cells that mediate the pupil light reflex has provided new insights into how the pupil responds to different properties of light. These ganglion cells are unique in their ability to transduce light into electrical energy. There are parallels between the electrophysiologic behavior of these cells in primates and the clinical pupil response to chromatic stimuli. Under photopic conditions, a red light stimulus produces a pupil constriction mediated predominantly by cone input via trans-synaptic activation of melanopsin-expressing retinal ganglion cells, whereas a blue light stimulus at high intensity produces a steady-state pupil constriction mediated primarily by direct intrinsic photoactivation of the melanopsin-expressing ganglion cells. Preliminary data in humans suggest that under photopic conditions, cones primarily drive the transient phase of the pupil light reflex, whereas intrinsic activation of the melanopsin-expressing ganglion cells contributes heavily to sustained pupil constriction. The use of chromatic light stimuli to elicit transient and sustained pupil light reflexes may become a clinical pupil test that allows differentiation between disorders affecting photoreceptors and those affecting retinal ganglion cells.

Chromatic pupillometry in patients with retinitis pigmentosa.

OBJECTIVE:: To evaluate the chromatic pupillary response as a means of assessing outer and inner retinal function in patients with retinitis pigmentosa (RP). DESIGN:: Evaluation of diagnostic technology. PARTICIPANTS:: Thirty-two patients with RP and visual loss and 43 normal subjects. METHODS:: Patients were tested with a chromatic pupillometer using red and blue lights (1, 10, and 100 cd/m(2)), and their pupil responses were compared with those from 43 normal subjects (reported previously). Visual field and electroretinography (ERG) results were examined and compared with the pupil responses. MAIN OUTCOME MEASURES:: The percent pupil contraction of the transient response to a low-intensity (1 cd/m(2)) blue light and high-intensity (100 cd/m(2)) red light and the sustained response to a high-intensity blue light was calculated for 1 eye of each subject. RESULTS:: The pupil responses to red and blue light at all intensities were recordable in all patients except 1, whose pupil responded only to bright blue light. There was a significant difference of the pupil response between patients with RP and normal subjects in testing conditions that emphasized rod (1 cd/m(2) blue light) or cone (100 cd/m(2) red light) contribution (P<0.001). Patients with a non-recordable scotopic ERG showed significantly reduced pupil responses (P<0.001) to low-intensity blue light (1 cd/m(2)). Patients with a non-recordable or abnormal photopic ERG showed significantly reduced pupil responses (P<0.05) to high-intensity red light (100 cd/m(2)). Patients with a nonrecordable ERG had the most visual field loss and reduced pupil responses. Unexpectedly, patients with RP showed a slower re-dilation of the pupil after termination of bright blue light compared with red light, a pattern not observed in normal subjects. CONCLUSIONS:: Pupil responses to red and blue light stimuli weighted to favor cone or rod input are significantly reduced in patients with RP but are still recordable in patients having a non-recordable ERG. In addition, outer photoreceptor disease appears to unmask a post-illumination pupillary constriction to bright blue light, most likely mediated by intrinsic activation of melanopsin ganglion cells. Chromatic pupillometry provides a novel, noninvasive method for following retinal functional status, particularly in patients with severe RP and non-recordable ERG. FINANCIAL DISCLOSURE(S):: Proprietary or commercial disclosure may be found after the references.

Exploring the rate-limiting steps in visual phototransduction recovery by bottom-up kinetic modeling

Phototransduction in vertebrate photoreceptor cells represents a paradigm of signaling pathways mediated by G-protein-coupled receptors (GPCRs), which share common modules linking the initiation of the cascade to the final response of the cell. In this work, we focused on the recovery phase of the visual photoresponse, which is comprised of several interacting mechanisms. We employed current biochemical knowledge to investigate the response mechanisms of a comprehensive model of the visual phototransduction pathway. In particular, we have improved the model by implementing a more detailed representation of the recoverin (Rec)-mediated calcium feedback on rhodopsin kinase and including a dynamic arrestin (Arr) oligomerization mechanism. The model was successfully employed to investigate the rate limiting steps in the recovery of the rod photoreceptor cell after illumination. Simulation of experimental conditions in which the expression levels of rhodospin kinase (RK), of the regulator of the G-protein signaling (RGS), of Arr and of Rec were altered individually or in combination revealed severe kinetic constraints to the dynamics of the overall network. Our simulations confirm that RGS-mediated effector shutdown is the rate-limiting step in the recovery of the photoreceptor and show that the dynamic formation and dissociation of Arr homodimers and homotetramers at different light intensities significantly affect the timing of rhodopsin shutdown. The transition of Arr from its oligomeric storage forms to its monomeric form serves to temper its availability in the functional state. Our results may explain the puzzling evidence that overexpressing RK does not influence the saturation time of rod cells at bright light stimuli. The approach presented here could be extended to the study of other GPCR signaling pathways.

A unique role for Kv3 voltage-gated potassium channels in starburst amacrine cell signaling in mouse retina

Direction-selective retinal ganglion cells show an increased activity evoked by light stimuli moving in the preferred direction. This selectivity is governed by direction-selective inhibition from starburst amacrine cells occurring during stimulus movement in the opposite or null direction. To understand the intrinsic membrane properties of starburst cells responsible for direction-selective GABA release, we performed whole-cell recordings from starburst cells in mouse retina. Voltage-clamp recordings revealed prominent voltage-dependent K+ currents. The currents were mostly blocked by 1 mm TEA, activated rapidly at voltages more positive than -20 mV, and deactivated quickly, properties reminiscent of the currents carried by the Kv3 subfamily of K+ channels. Immunoblots confirmed the presence of Kv3.1 and Kv3.2 proteins in retina and immunohistochemistry revealed their expression in starburst cell somata and dendrites. The Kv3-like current in starburst cells was absent in Kv3.1-Kv3.2 knock-out mice. Current-clamp recordings showed that the fast activation of the Kv3 channels provides a voltage-dependent shunt that limits depolarization of the soma to potentials more positive than -20 mV. This provides a mechanism likely to contribute to the electrical isolation of individual starburst cell dendrites, a property thought essential for direction selectivity. This function of Kv3 channels differs from that in other neurons where they facilitate high-frequency repetitive firing. Moreover, we found a gradient in the intensity of Kv3.1b immunolabeling favoring proximal regions of starburst cells. We hypothesize that this Kv3 channel gradient contributes to the preference for centrifugal signal flow in dendrites underlying direction-selective GABA release from starburst amacrine cells.

Differential monocular vs. binocular pupil responses from melanopsin-based photoreception in patients with anterior ischemic optic neuropathy.

We examined the effect of anterior ischemic optic neuropathy (AION) on the activity of intrinsically photosensitive retinal ganglion cells (ipRGCs) using the pupil as proxy. Eighteen patients with AION (10 unilateral, 8 bilateral) and 29 age-matched control subjects underwent chromatic pupillometry. Red and blue light stimuli increasing in 0.5 log steps were presented to each eye independently under conditions of dark and light adaptation. The recorded pupil contraction was plotted against stimulus intensity to generate scotopic and photopic response curves for assessment of synaptically-mediated ipRGC activity. Bright blue light stimuli presented monocularly and binocularly were used for melanopsin activation. The post-stimulus pupil size (PSPS) at the 6th second following stimulus offset was the marker of intrinsic ipRGC activity. Finally, questionnaires were administered to assess the influence of ipRGCs on sleep. The pupil response and PSPS to all monocularly-presented light stimuli were impaired in AION eyes, indicating ipRGC dysfunction. To binocular light stimulation, the PSPS of AION patients was similar to that of controls. There was no difference in the sleep habits of the two groups. Thus after ischemic injury to one or both optic nerves, the summated intrinsic ipRGC activity is preserved when both eyes receive adequate light exposure.

Reconsiderations about the clinical importance of the sympathetic skin response

Over the last few years, neurologists have been showing increasing interest in the study of the sympathetic skin response (SSR). In the present report we describe a simple method that permitted us to determine a wide variation of SSR in response to different stimuli such as respiration, deglutition, blinking, skeletal movements, biting, auditory or light stimuli, vocalization, and sphincter contraction. These results raise doubts about the role of SSR as a complementary diagnostic method.

Estruturas do olho do Macrobrachium amazonicum (Heller, 1862) (Crustacea, Decapoda, Palaemonidae): estudo utilizando microscopia óptica e de varredura.

O gênero Macrobrachium contém mais de 120 espécies e ocorre nas regiões tropicais e subtropicais de todo o mundo (VALENTI, 1987). São camarões de água doce da família Palaemonidae e da ordem Decapoda (RAFINESQUE, 1815; LATREILLER, 1802). No Brasil existem 18 espécies, até agora classificadas, distribuídas ao longo da bacia amazônica (MELO 2003). Entre estas, o Macrobrachium amazonicum (HELLER, 1862) conhecido popularmente como camarão-sossego ou camarão-canela, amplamente empregados na carcinicultura. Os crustáceos dispõem de estruturas sensitivas localizadas no cefalotórax, que permitem receber estímulos do meio para localizar e capturar o alimento (BARNES, 1998). Os olhos compostos estão presentes em todas as classes de crustáceo. Assim, a hipótese levantada nessa pesquisa foi que no Macrobrachium amazonicum, estes olhos são do tipo de superposição reflexiva, onde o aparelho dióptrico e o rabdômero se estendem em camadas e está separado por uma zona clara não pigmentada. Neste trabalho temos como objetivos: Avaliar os aspectos morfológicos do olho do Macrobrachium amazonicum em microscopia eletrônica de varredura; Caracterizar a morfologia das células fotorreceptoras; Descrever as estruturas morfológicas do olho do M. amazonicum; Caracterizar as relações morfométricas entre o olho e as demais estruturas do M. amazonicum. Os animais foram adquiridos no distrito de Mosqueiro nos períodos de chuvas, março de 2009 e março de 2010, com pescadores no município de Santa Bárbara, área metropolitana de Belém, e transportados para o laboratório em caixas de isopor, sendo mantidos em quarentena em um recipiente contendo uma solução de permanganato de potássio a 1,3 mg/L (CARNEIRO et al., 005). Os animais foram fixados em Davidson e Karnovisky, em seguida os olhos de cada animal foram cuidadosamente seccionados e colocados em frascos plásticos. Nas relações biométricas foram realizadas a análise de variância com α = 0,05, foi realizada com Bio Estat 5.0 para os comprimentos do olho látero-lateral e ântero-posterior dos quatro morfotipos, e SigmaPlot 11.0 e regressão linear simples, para as variáveis olho total e cefalotórax. Observou-se que o tamanho médio do cefalotórax, do corpo, do olho é respectivamente: 21,03 mm; 70,62 mm e 4,52 mm, sendo que, o peso médio do camarão foi de 7,97 g. Os valores máximos registrados dessas estruturas foram de 31,95 mm para o cefalotórax; 100,10 mm para o tamanho do corpo; 6,80 mm para o tamanho do olho e de 20,54 g para o peso do camarão. Após análise histológica foram identificadas as seguintes estruturas (Figs. 13 e 14 A-B): córnea, cone cristalino, pigmento distal, haste do cone, zona clara, cutícula, retina, rabdoma, fibras do nervo óptico e lamina. Com relação à microscopia eletrônica de varredura, foram selecionadas para observação as principais estruturas e especialmente o arranjo em seção transversal quadrada dos omatídeos (Figs. 16 A-B). As análises morfométrica e morfológica (por histologia e microscopia eletrônica de varredura) apontaram características próprias de Macrobrachium amazonicum adultos oriundos da região metropolitana de Belém. Estes achados incluem uma óptica de superposição reflexiva com olhos adaptados a percepção de estímulos luminosos.

A Schiff base connectivity switch in sensory rhodopsin signaling.

Sensory rhodopsin I (SRI) in Halobacterium salinarum acts as a receptor for single-quantum attractant and two-quantum repellent phototaxis, transmitting light stimuli via its bound transducer HtrI. Signal-inverting mutations in the SRI-HtrI complex reverse the single-quantum response from attractant to repellent. Fast intramolecular charge movements reported here reveal that the unphotolyzed SRI-HtrI complex exists in two conformational states, which differ by their connection of the retinylidene Schiff base in the SRI photoactive site to inner or outer half-channels. In single-quantum photochemical reactions, the conformer with the Schiff base connected to the cytoplasmic (CP) half-channel generates an attractant signal, whereas the conformer with the Schiff base connected to the extracellular (EC) half-channel generates a repellent signal. In the wild-type complex the conformer equilibrium is poised strongly in favor of that with CP-accessible Schiff base. Signal-inverting mutations shift the equilibrium in favor of the EC-accessible Schiff base form, and suppressor mutations shift the equilibrium back toward the CP-accessible Schiff base form, restoring the wild-type phenotype. Our data show that the sign of the behavioral response directly correlates with the state of the connectivity switch, not with the direction of proton movements or changes in acceptor pK(a). These findings identify a shared fundamental process in the mechanisms of transport and signaling by the rhodopsin family. Furthermore, the effects of mutations in the HtrI subunit of the complex on SRI Schiff base connectivity indicate that the two proteins are tightly coupled to form a single unit that undergoes a concerted conformational transition.