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.

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 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.

Interphotoreceptor coupling: an evolutionary perspective.

In the vertebrate retina, signals generated by cones of different spectral preference and by highly sensitive rod photoreceptors interact at various levels to extract salient visual information. The first opportunity for such interaction is offered by electrical coupling of the photoreceptors themselves, which is mediated by gap junctions located at the contact points of specialised cellular processes: synaptic terminals, telodendria and radial fins. Here, we examine the evolutionary pressures for and against interphotoreceptor coupling, which are likely to have shaped how coupling is deployed in different species. The impact of coupling on signal to noise ratio, spatial acuity, contrast sensitivity, absolute and increment threshold, retinal signal flow and colour discrimination is discussed while emphasising available data from a variety of vertebrate models spanning from lampreys to primates. We highlight the many gaps in our knowledge, persisting discrepancies in the literature, as well as some major unanswered questions on the actual extent and physiological role of cone-cone, rod-cone and rod-rod communication. Lastly, we point toward limited but intriguing evidence suggestive of the ancestral form of coupling among ciliary photoreceptors.

Gender and age normalization and ventilation efficiency during exercise in heart failure with reduced ejection fraction.

AIMS: Ventilation vs. carbon dioxide production (VE/VCO2 ) is among the strongest cardiopulmonary exercise testing prognostic parameters in heart failure (HF). It is usually reported as an absolute value. The current definition of normal VE/VCO2 slope values is inadequate, since it was built from small groups of subjects with a particularly limited number of women and elderly. We aimed to define VE/VCO2 slope prediction formulas in a sizable population and to test whether the prognostic power of VE/VCO2 slope in HF was different if expressed as a percentage of the predicted value or as an absolute value. METHODS AND RESULTS: We calculated the linear regressions between age and VE/VCO2 slope in 1136 healthy subjects (68% male, age 44.9 ± 14.5, range 13-83 years). We then applied age-adjusted and sex-adjusted formulas to predict VE/VCO2 slope to HF patients included in the metabolic exercise test data combined with cardiac and kidney indexes score database, which counts 6112 patients (82% male, age 61.4 ± 12.8, left ventricular ejection fraction 33.2 ± 10.5%, peakVO2 14.8 ± 4.9, mL/min/kg, VE/VCO2 slope 32.7 ± 7.7) from 24 HF centres. Finally, we evaluated whether the use of absolute values vs. percentages of predicted VE/VCO2 affected HF prognosis prediction (composite of cardiovascular mortality + urgent transplant or left ventricular assist device). We did so in the entire cardiac and kidney indexes score population and separately in HF patients with severe (peakVO2 < 14 mL/min/kg, n = 2919, 61.1 events/1000 pts/year) or moderate (peakVO2 ≥ 14 mL/min/kg, n = 3183, 19.9 events/1000 pts/year) HF. In the healthy population, we obtained the following equations: female, VE/VCO2 = 0.052 × Age + 23.808 (r = 0.192); male, VE/VCO2 = 0.095 × Age + 20.227 (r = 0.371) (P = 0.007). We applied these formulas to calculate the percentages of predicted VE/VCO2 values. The 2-year survival prognostic power of VE/VCO2 slope was strong, and it was similar if expressed as absolute value or as a percentage of predicted value (AUCs 0.686 and 0.690, respectively). In contrast, in severe HF patients, AUCs significantly differed between absolute values (0.637) and percentages of predicted values (0.650, P = 0.0026). Moreover, VE/VCO2 slope expressed as a percentage of predicted value allowed to reclassify 6.6% of peakVO2 < 14 mL/min/kg patients (net reclassification improvement = 0.066, P = 0.0015). CONCLUSIONS: The percentage of predicted VE/VCO2 slope value strengthens the prognostic power of VE/VCO2 in severe HF patients, and it should be preferred over the absolute value for HF prognostication. Furthermore, the widespread use of VE/VCO2 slope expressed as percentage of predicted value can improve our ability to identify HF patients at high risk, which is a goal of utmost clinical relevance.

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, ±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.

Lumbar spinal cord neurons putatively involved in ejaculation are sexually dimorphic in early postnatal mice.

A crucial role in ejaculation is thought to be played by a population of lumbar spino-thalamic neurons (LSt), which express galanin and other neuropeptides. In rats, these neurons are activated with ejaculation and their lesion selectively abolishes ejaculation but not other mating behaviors. Consistently with their role, in adult rats and humans, LSt neurons are sexually dimorphic, being more numerous in males. Here we examined whether sexual dimorphism arises early in development, using a transgenic mouse line in which the expression of fluorescent protein is driven by the galanin promoter. We focused on postnatal day 4, shortly after a transient perinatal androgen surge in males that could play an organizational role in LSt development. We found a population of brightly fluorescent neurons organized in bilateral columns dorsolateral to the central canal in segments L1-L5, the expected location of the LSt group. Their number was close to that of adult preparations and significantly greater in male than in female siblings (+19%; CI95% : +13% to +27%; p < .01). This was not due to a generalized higher galanin expression in the male since fluorescent L4 DRG neurons, innervating the hindlimbs and lower back, were not significantly dimorphic (-4%; CI95% : -10% to +8%; p = .92). Unexpectedly, we found in cervical segments a population of fluorescent neurons having a location relative to the central canal similar to the LSt. Thus, the LSt group is sexually dimorphic soon after birth. However, it is possible that only a subset of its neurons participate in the control of ejaculation.

Two simple criteria to estimate an objective's performance when imaging in non design tissue clearing solutions.

Tissue 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.

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.

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.

Loss of HCN1 enhances disease progression in mouse models of CNG channel-linked retinitis pigmentosa and achromatopsia.

Most inherited blinding diseases are characterized by compromised retinal function and progressive degeneration of photoreceptors. However, the factors that affect the life span of photoreceptors in such degenerative retinal diseases are rather poorly understood. Here, we explore the role of hyperpolarization-activated cyclic nucleotide-gated channel 1 (HCN1) in this context. HCN1 is known to adjust retinal function under mesopic conditions, and although it is expressed at high levels in rod and cone photoreceptor inner segments, no association with any retinal disorder has yet been found. We investigated the effects of an additional genetic deletion of HCN1 on the function and survival of photoreceptors in a mouse model of CNGB1-linked retinitis pigmentosa (RP). We found that the absence of HCN1 in Cngb1 knockout (KO) mice exacerbated photoreceptor degeneration. The deleterious effect was reduced by expression of HCN1 using a viral vector. Moreover, pharmacological inhibition of HCN1 also enhanced rod degeneration in Cngb1 KO mice. Patch-clamp recordings revealed that the membrane potentials of Cngb1 KO and Cngb1/Hcn1 double-KO rods were both significantly depolarized. We also found evidence for altered calcium homeostasis and increased activation of the protease calpain in Cngb1/Hcn1 double-KO mice. Finally, the deletion of HCN1 also exacerbated degeneration of cone photoreceptors in a mouse model of CNGA3-linked achromatopsia. Our results identify HCN1 as a major modifier of photoreceptor degeneration and suggest that pharmacological inhibition of HCN channels may enhance disease progression in RP and achromatopsia patients.

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.

Effective delivery of recombinant proteins to rod photoreceptors via lipid nanovesicles.

The potential of liposomes to deliver functional proteins in retinal photoreceptors and modulate their physiological response was investigated by two experimental approaches. First, we treated isolated mouse retinas with liposomes encapsulating either recoverin, an important endogenous protein operating in visual phototransduction, or antibodies against recoverin. We then intravitrally injected in vivo liposomes encapsulating either rhodamin B or recoverin and we investigated the distribution in retina sections by confocal microscopy. The content of liposomes was found to be released in higher amount in the photoreceptor layer than in the other regions of the retina and the functional effects of the release were in line with the current model of phototransduction. Our study sets the basis for quantitative investigations aimed at assessing the potential of intraocular protein delivery via biocompatible nanovesicles, with promising implications for the treatment of retinal diseases affecting the photoreceptor layer.

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. DOI: http://dx.doi.org/10.7554/eLife.01386.001.

Detecting single photons: a supramolecular matter?

Rod photoreceptors detect single photons through a tradeoff of light collecting ability, amplification and speed. Key roles are played by rhodopsin (Rh) and transducin (G(t)), whose complex supramolecular organization in outer segment disks begs for a functional interpretation. Here we review past and recent evidence of a temperature-dependence of photon detection by mammalian rods, and link this phenomenon with the putative oligomeric organization of Rh and new ideas on the dynamics of Rh-G(t) interaction. Identifying an electrophysiological correlate of the supramolecular organization of Rh and G(t) may shed light on the evolutionary advantage it confers to night vision.

The photovoltage of rods and cones in the dark-adapted mouse retina.

Research on photoreceptors has led to important insights into how light signals are detected and processed in the outer retina. Most information about photoreceptor function, however, comes from lower vertebrates. The large majority of mammalian studies are based on suction pipette recordings of outer segment currents, a technique that doesn't allow examination of phenomena occurring downstream of phototransduction. Only a small number of whole-cell recordings have been made, mainly in the macaque. Due to the growing importance of the mouse in vision research, we have optimized a retinal slice preparation that allows the reliable collection of perforated-patch recordings from light responding rods and cones. Unexpectedly, the frequency of cone recordings was much higher than their numeric proportion of ∼3%. This allowed us to obtain direct functional evidence suggestive of rod­cone coupling in the mouse. Moreover, rods had considerably larger single photon responses than previously published for mammals (3.44 mV, SD 1.37, n = 19 at 24°C; 2.46 mV, SD 1.08, n = 10 at 36°C), and a relatively high signal/noise ratio (6.4, SD 1.8 at 24°C; 6.8, SD 2.8 at 36°C). Both findings imply a more favourable transmission at the rod­rod bipolar cell synapse. Accordingly, relatively few photoisomerizations were sufficient to elicit a half-maximal response (6.7, SD 2.7, n = 5 at 24°C; 10.6, SD 1.7, n = 3 at 36°C), leading to a narrow linear response range. Our study demonstrates new features of mammalian photoreceptors and opens the way for further investigations into photoreceptor function using retinas from mutant mouse models.

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 I(h) show that, contrary to previous reports, I(kx) alone is able to generate the fast initial transient in the rod bright flash response. Here we demonstrate that the relative contribution of I(h) and I(kx) 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.

Neuronal substrates for state-dependent changes in coordination between motoneuron pools during fictive locomotion in the lamprey spinal cord.

Locomotion relies on a precisely timed activation of sets of motoneurons. A fundamental question is how this is achieved. In the lamprey, fin and myotomal motoneurons located on the same side of the spinal cord display alternating activity during straight swimming. The neural mechanism underlying this alternation is studied here during fictive locomotion induced by superfusion with NMDA, or locomotor bursting induced by electrical stimulation. If the spinal cord is split longitudinally, each hemicord still displays rhythmic locomotor related burst activity, but now fin and myotomal motoneurons become active in-phase. The out-of-phase activation of fin motoneurons persists only when at least three segments are left intact in the rostral part of the spinal cord. Proper coordination of fin motoneurons thus requires input from contralateral rostral segments. We show that commissural excitatory interneurons with long descending axons, previously reported to be active in phase with their ipsilateral myotomal motoneurons, provide monosynaptic excitation to contralateral fin motoneurons. Together, these results strongly indicate that, although myotomal motoneurons receive their phasic excitation from ipsilateral excitatory interneurons, fin motoneurons are mainly driven from the contralateral segmental network during bilateral locomotor activity. However, during unilateral bursting, fin and myotomal motoneurons instead receive a common input, which is apparently masked during normal fictive swimming. The spinal organization thus also provides circuitry for different patterns of coordination, i.e., alternation or coactivation of the two pools of motoneurons, which may subserve different forms of locomotor behavior.