Versatile bipolar temperature controller for custom in vitro applications

Effective 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, \ub15.0 A), sufficient to bring a well designed holder for standard 35 mm chambers from 23 \ub0C up to 37 \ub0C in ~1 min and down to 3 \ub0C in ~4 min. Any biologist with some technical prowess should be able to follow our instructions from modeling to assembly and calibration

An ex vivo electroretinographic apparatus for the ml-scale testing of drugs to one day and beyond

When 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

A cambrian origin for vertebrate rods

Vertebrates 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

Connexin 36 expression is required for electrical coupling between mouse rods and cones.

Rod-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.We thank Drs. Klaus Willecke and Karin Dedek for generously donating Cx36^ +/del[LacZ] founder mice to establish a colony in Pisa. Intramural funding was provided by the University of Pisa to L.C. and C.G. and by the Ministero dell’Istruzione, dell’Università e della Ricerca (2010599KBR_003) to C.G

Mouse rods signal through gap junctions with cones

Rod 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

A hybrid stochastic/deterministic model of single photon response and light adaptation in mouse rods

The phototransduction cascade is paradigmatic for signaling pathways initiated by G protein-coupled receptors and is characterized by a fine regulation of photoreceptor sensitivity and electrical response to a broad range of light stimuli. Here, we present a biochemically comprehensive model of phototransduction in mouse rods based on a hybrid stochastic and deterministic mathematical framework, and a quantitatively accurate description of the rod impedance in the dark. The latter, combined with novel patch clamp recordings from rod outer segments, enables the interconversion of dim flash responses between photovoltage and photocurrent and thus direct comparison with the simulations. The model reproduces the salient features of the experimental photoresponses at very dim and bright stimuli, for both normal photoreceptors and those with genetically modified cascade components. Our modelling approach recapitulates a number of recent findings in vertebrate phototransduction. First, our results are in line with the recently established requirement of dimeric activation of PDE6 by transducin and further show that such conditions can be fulfilled at the expense of a significant excess of G protein activated by rhodopsin. Secondly, simulations suggest a crucial role of the recoverin-mediated Ca2+-feedback on rhodopsin kinase in accelerating the shutoff, when light flashes are delivered in the presence of a light background. Finally, stochastic simulations suggest that transient complexes between dark rhodopsin and transducin formed prior to light stimulation increase the reproducibility of single photon responses. Current limitations of the model are likely associated with the yet unknown mechanisms governing the shutoff of the cascade

Recombinant protein delivery enables modulation of the phototransduction cascade in mouse retina

Inherited retinal dystrophies are often associated with mutations in the genes involved in the phototransduction cascade in photoreceptors, a paradigmatic signaling pathway mediated by G protein-coupled receptors. Photoreceptor viability is strictly dependent on the levels of the second messengers cGMP and Ca2+. Here we explored the possibility of modulating the phototransduction cascade in mouse rods using direct or liposome-mediated administration of a recombinant protein crucial for regulating the interplay of the second messengers in photoreceptor outer segments. The effects of administration of the free and liposome-encapsulated human guanylate cyclase-activating protein 1 (GCAP1) were compared in biological systems of increasing complexity (in cyto, ex vivo, and in vivo). The analysis of protein biodistribution and the direct measurement of functional alteration in rod photoresponses show that the exogenous GCAP1 protein is fully incorporated into the mouse retina and photoreceptor outer segments. Furthermore, only in the presence of a point mutation associated with cone-rod dystrophy in humans p.(E111V), protein delivery induces a disease-like electrophysiological phenotype, consistent with constitutive activation of the retinal guanylate cyclase. Our study demonstrates that both direct and liposome-mediated protein delivery are powerful complementary tools for targeting signaling cascades in neuronal cells, which could be particularly important for the treatment of autosomal dominant genetic diseases

L’ecografia polmonare per il pediatra

L’ecografia polmonare si sta dimostrando una metodica di grande interesse e negli ultimi anni si è assistito ad un fiorire di pubblicazioni sull’argomento. In particolar modo, la sua semplicità d’uso e d’apprendimento ne stanno garantendo una forte diffusione. Se paragonata poi alla radiografia del torace, la più diffusa indagine di imaging in pneumologia pediatrica, l’ecografia polmonare appare molto promettente. In questo articolo ci prefiggiamo di fornire alcune informazioni basilari sull’ecografia polmonare che possono però diventare un importante spunto per la comprensione di questa innovativa metodica.The lung ultrasound is object of a strong interest and in the lasts years there has been a huge increase in the number of publications on the topic. Its simplicity of use and learning are ensuring it an increasingly widespread use. If compared to the chest radiograph, the most used imaging technique in pediatric pulmonology, lung ultrasound appears very promising. In this article we aim to provide some basic information on lung ultrasound that can become an important start point for understanding this innovative technique

MiR-211 is essential for adult cone photoreceptor maintenance and visual function.

MicroRNAs (miRNAs) are key post-transcriptional regulators of gene expression that play an important role in the control of fundamental biological processes in both physiological and pathological conditions. Their function in retinal cells is just beginning to be elucidated, and a few have been found to play a role in photoreceptor maintenance and function. MiR-211 is one of the most abundant miRNAs in the developing and adult eye. However, its role in controlling vertebrate visual system development, maintenance and function so far remain incompletely unexplored. Here, by targeted inactivation in a mouse model, we identify a critical role of miR-211 in cone photoreceptor function and survival. MiR-211 knockout (-/-) mice exhibited a progressive cone dystrophy accompanied by significant alterations in visual function. Transcriptome analysis of the retina from miR-211-/- mice during cone degeneration revealed significant alteration of pathways related to cell metabolism. Collectively, this study highlights for the first time the impact of miR-211 function in the retina and significantly contributes to unravelling the role of specific miRNAs in cone photoreceptor function and survival

Processing of Retinal Signals in Normal and HCN Deficient Mice

This study investigates the role of two different HCN channel isoforms in the light response of the outer retina. Taking advantage of HCN-deficient mice models and of in vitro (patch-clamp) and in vivo (ERG) recordings of retinal activity we show that HCN1 and HCN2 channels are expressed at distinct retinal sites and serve different functions. Specifically, HCN1 operate mainly at the level of the photoreceptor inner segment from where, together with other voltage sensitive channels, they control the time course of the response to bright light. Conversely, HCN2 channels are mainly expressed on the dendrites of bipolar cells and affect the response to dim lights. Single cell recordings in HCN1−/− mice or during a pharmacological blockade of Ih show that, contrary to previous reports, Ikx alone is able to generate the fast initial transient in the rod bright flash response. Here we demonstrate that the relative contribution of Ih and Ikx to the rods' temporal tuning depends on the membrane potential. This is the first instance in which the light response of normal and HCN1- or HCN2-deficient mice is analyzed in single cells in retinal slice preparations and in integrated full field ERG responses from intact animals. This comparison reveals a high degree of correlation between single cell current clamp data and ERG measurements. A novel picture emerges showing that the temporal profile of the visual response to dim and bright luminance changes is separately determined by the coordinated gating of distinct voltage dependent conductances in photoreceptors and bipolar cells