1,720,976 research outputs found
Interazioni tra fotorecettori
Full text linkBastoncelli e coni sono in grado di comunicare tra loro a livello elettrico e chimico mediante piccoli pori (connessoni). I coni possono diventare più sensibili alla luce se funzionalmente accoppiati ai bastoncelli
Slow light response kinetics in rods points towards a perturbation of the normal cellular milieu
Full text linkAn Ex Vivo Electroretinographic Apparatus for the mL-Scale Testing of Drugs to One Day and Beyond
Full text linkWhen screening new drugs to treat retinal diseases, ex vivo electroretinography (ERG) potentially combines the experimental throughput of its traditional in vivo counterpart, with greater mechanistic insight and reproducible delivery. To date, this technique was used in experiments with open loop superfusion and lasting up to a few hours. Here, we present a compact apparatus that provides continuous and simultaneous recordings of the scotopic a-waves from four mouse retinas for much longer durations. Crucially, each retina can be incubated at 37 °C in only 2 mL of static medium, enabling the testing of very expensive drugs or nano devices. Light sensitivity and response kinetics of these preparations remain in the physiological range throughout incubation, displaying only very slow drifts. As an example application, we showed that barium, a potassium channel blocker used to abolish the glial component of the ERG, displayed no overt side effects on photoreceptors over several hours. In another example, we fully regenerated a partially bleached retina using a minimal quantity of 9-cis-retinal. Finally, we demonstrated that including antibiotic in the incubation medium extends physiological light responses to over one day. This system represents a necessary stepping stone towards the goal of combining ERG recordings with organotypically cultured retinas
Versatile bipolar temperature controller for custom in vitro applications
Full text linkEffective temperature control is crucial in many studies of isolated biological tissues, with preparations often requiring specialized holding chambers. In these situations, the design flexibility and optimizations offered by a custom made temperature controller may be preferable over a commercial model. We present a versatile controller for heating and cooling applications, providing simple step-by-step instructions to mathematically model your specific system and optimize controller parameters. The apparatus uses analog components and linear stages to simplify circuit comprehension and customization, achieving fast transitions with small static errors and overshoots over a wide range of temperatures without readjustment. A fully featured rackable enclosure is complemented by two temperature probes based on the LMT70A linear microchip sensor (for the control loop and for bath monitoring). BNC outputs provide scaled probe signals for continuous temperature data acquisition. The maximum achievable power output of the controller is -23.5 W/+22.0 W (-4.7 V/+4.4 V, ±5.0 A), sufficient to bring a well designed holder for standard 35 mm chambers from 23 °C up to 37 °C in ~1 min and down to 3 °C in ~4 min. Any biologist with some technical prowess should be able to follow our instructions from modeling to assembly and calibration
Two simple criteria to estimate an objective's performance when imaging in non design tissue clearing solutions
Full text linkTissue clearing techniques are undergoing a renaissance motivated by the need to image fluorescent neurons, and other cells, deep in the sample without physical sectioning. Optical transparency is achieved by equilibrating tissues with high refractive index (RI) solutions. When the microscope objective is not perfectly matched to the RI of the cleared sample, aberrations are introduced. We present two simple-to-calculate numerical criteria predicting: (i) the degradation in image quality (brightness and resolution) from optimal conditions of any clearing solution/objective combination; (ii) which objective, among several available, achieves the highest resolution in a given medium. We derived closed form approximations for image quality degradation versus RI mismatch and other parameters available to the microscopist, validated them with computed and measured aberrated point spread functions and by imaging fluorescent neurons in high RI solution. These approximations apply to the widefield fluorescent microscope but are also relevant to more advanced microscopes. Currently, to accurately predict the impact of RI mismatch-induced aberrations on imaging, the life scientist must examine theoretical or experimental point spread functions (PSFs) obtained under the optical configuration of interest. These criteria can be used to select a suitable objective for the chosen clearing method (particularly when subject to budget constraints) or to tweak a clearing solution RI to the available objectives. Even with a nominally optimal objective, one may wish to assess the impact of any small unavoidable mismatches
Mouse rods signal through gap junctions with cones
Full text linkRod and cone photoreceptors are coupled by gap junctions (GJs), relatively large channels able to mediate both electrical and molecular communication. Despite their critical location in our visual system and evidence that they are dynamically gated for dark/light adaptation, the full impact that rod–cone GJs can have on cone function is not known. We recorded the photovoltage of mouse cones and found that the initial level of rod input increased spontaneously after obtaining intracellular access. This process allowed us to explore the underlying coupling capacity to rods, revealing that fully coupled cones acquire a striking rod-like phenotype. Calcium, a candidate mediator of the coupling process, does not appear to be involved on the cone side of the junctional channels. Our findings show that the anatomical substrate is adequate for rod–cone coupling to play an important role in vision and, possibly, in biochemical signaling among photoreceptors
Connexin 36 expression is required for electrical coupling between mouse rods and cones
Full text linkRod-cone gap junctions mediate the so-called “secondary rod pathway”, one of three routes that convey rod photoreceptor signals across the retina. Connexin 36 (Cx36) is expressed at these gap junctions, but an unidentified connexin protein also seems to be expressed. Cx36 knockout mice have been used extensively in the quest to dissect the roles in vision of all three pathways, with the assumption, never directly tested, that rod-cone electrical coupling is abolished by deletion of this connexin isoform. We previously showed that when wild type mouse cones couple to rods, their apparent dynamic range is extended toward lower light intensities, with the appearance of large responses to dim flashes (up to several mV) originating in rods. Here we recorded from the cones of Cx36del[LacZ]/del[LacZ] mice and found that dim flashes of the same intensity evoked at most small sub-millivolt responses. Moreover, these residual responses originated in the cones themselves, since: (i) their spectral preference matched that of the recorded cone and not of rods, (ii) their time-to-peak was shorter than in coupled wild type cones, (iii) a pharmacological block of gap junctions did not reduce their amplitude. Taken together, our data show that rod signals are indeed absent in the cones of Cx36 knockout mice. This study is the first direct demonstration that Cx36 is crucial for the assembly of functional rod-cone gap junctional channels, implying that its genetic deletion is a reliable experimental approach to eliminate rod-cone coupling
Rod-cone coupling studied in single mouse cones: impact and regulation
No full textPurpose:Rod-cone gap junctions (GJs) in mammals are thought of being an escape route for rod signals when incoming light is greater than a few sparse photons and the rod-rod bipolar synapse is saturated. While indirect evidence suggest that this pathway is dynamically regulated by light and circadian rhythm, the only direct functional tests so far, done in Macaque, have found rod-cone coupling to be static. We optimized patch recordings from mouse cones to investigate the impact and regulatory latitude of rod-cone coupling.
Methods:Cones (n=55) were recorded with perforated patch-clamp in retinal slices from wt mice, and from animals lacking connexin isoform 36 (Cx36-KO). Photovoltage responses were evoked with green/UV stimulation protocols, consisting of dim (16.6 ph/μm2) and bright (1570 or 3140 ph/μm2) flashes, designed to dissect and quantify rod input through differences in light sensitivity, kinetics of recovery from saturation and spectral preference. Control recordings in rods were also performed.
Results:Most wt cones expressed rod-like features, including: (1) responses to dim flashes (> 1 mV in 23 of 42); (2) responses to bright flashes followed by slow plateaus and a transient suppression of dim flash responses; (3) slow recovery of dim flash responses and plateaus upon return to darkness after prolonged rod-saturating light background. While some cones expressed rod features at the beginning of the recording, most cones acquired them progressively starting from seal formation, with a time course of minutes. Irrespective of their intrinsic spectral type (G- or UV-preferring, as determined with rod-saturating pre-flashes) for dim flashes cones preferred G, in line with rods (n=15). Medium-large dim flash responses were observed in both M and S/M cones, while, of two S cones recorded, only one had a small response. Dim and bright flash responses had different reversal potentials, consistent with an origin in separate electrotonic compartments. The involvement of GJs was confirmed with the specific antagonist meclofenamic acid (100 μM), which abolished dim flash responses. In contrast to wt animals, in Cx36-KO mice cones did not appear to be able to couple to rods.
Conclusions:Mouse cones can couple remarkably well to rods and acquire a rod-like functional phenotype. Coupling strength varies among cones and is able to undergo rapid up-regulation triggered by perturbation of the intracellular milieu
A cambrian origin for vertebrate rods
Full text linkVertebrates acquired dim light vision when an ancestral cone evolved into the rod photoreceptor at an unknown stage preceding the last common ancestor of extant jawed vertebrates (~420 million years ago Ma). The jawless lampreys provide a unique opportunity to constrain the timing of this advance, as their line diverged ~505 Ma and later displayed high morphological stability. We recorded with patch electrodes the inner segment photovoltages and with suction electrodes the outer segment photocurrents of Lampetra fluviatilis retinal photoreceptors. Several key functional features of jawed vertebrate rods are present in their phylogenetically homologous photoreceptors in lamprey: crucially, the efficient amplification of the effect of single photons, measured by multiple parameters, and the flow of rod signals into cones. These results make convergent evolution in the jawless and jawed vertebrate lines unlikely and indicate an early origin of rods, implying strong selective pressure toward dim light vision in Cambrian ecosystems
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