In Intact Mammalian Photoreceptors, Ca2+-dependent Modulation of cGMP-gated Ion Channels Is Detectable in Cones but Not in Rods

In the mammalian retina, cone photoreceptors efficiently adapt to changing background light intensity and, therefore, are able to signal small differences in luminance between objects and backgrounds, even when the absolute intensity of the background changes over five to six orders of magnitude. Mammalian rod photoreceptors, in contrast, adapt very little and only at intensities that nearly saturate the amplitude of their photoresponse. In search of a molecular explanation for this observation we assessed Ca2+-dependent modulation of ligand sensitivity in cyclic GMP–gated (CNG) ion channels of intact mammalian rods and cones. Solitary photoreceptors were isolated by gentle proteolysis of ground squirrel retina. Rods and cones were distinguished by whether or not their outer segments bind PNA lectin. We measured membrane currents under voltage-clamp in photoreceptors loaded with Diazo-2, a caged Ca2+ chelator, and fixed concentrations of 8Br-cGMP. At 600 nM free cytoplasmic Ca2+ the midpoint of the cone CNG channels sensitivity to 8BrcGMP, 8BrcGMPK1/2, is ∼2.3 μM. The ligand sensitivity is less in rod than in cone channels. Instantly decreasing cytoplasmic Ca2+ to <30 nM activates a large inward membrane current in cones, but not in rods. Current activation arises from a Ca2+ -dependent modulation of cone CNG channels, presumably because of an increase in their affinity to the cyclic nucleotide. The time course of current activation is temperature dependent; it is well described by a single exponential process of ∼480 ms time constant at 20–21°C and 138 ms at 32°C. The absence of detectable Ca2+-dependent CNG current modulation in intact rods, in view of the known channel modulation by calmodulin in-vitro, affirms the modulation in intact rods may only occur at low Ca2+ concentrations, those expected at intensities that nearly saturate the rod photoresponse. The correspondence between Ca2+ dependence of CNG modulation and the ability to light adapt suggest these events are correlated in photoreceptors

The Na+/Ca2+, K+ exchanger NCKX4 is required for efficient cone-mediated vision

Calcium (Ca2+) plays an important role in the function and health of neurons. In vertebrate cone photoreceptors, Ca2+ controls photoresponse sensitivity, kinetics, and light adaptation. Despite the critical role of Ca2+ in supporting the function and survival of cones, the mechanism for its extrusion from cone outer segments is not well understood. Here, we show that the Na+/Ca2+, K+ exchanger NCKX4 is expressed in zebrafish, mouse, and primate cones. Functional analysis of NCKX4-deficient mouse cones revealed that this exchanger is essential for the wide operating range and high temporal resolution of cone-mediated vision. We show that NCKX4 shapes the cone photoresponse together with the cone-specific NCKX2: NCKX4 acts early to limit response amplitude, while NCKX2 acts late to further accelerate response recovery. The regulation of Ca2+ by NCKX4 in cones is a novel mechanism that supports their ability to function as daytime photoreceptors and promotes their survival

Adaptive Filtering Enhances Information Transmission in Visual Cortex

Sensory neuroscience seeks to understand how the brain encodes natural environments. However, neural coding has largely been studied using simplified stimuli. In order to assess whether the brain's coding strategy depend on the stimulus ensemble, we apply a new information-theoretic method that allows unbiased calculation of neural filters (receptive fields) from responses to natural scenes or other complex signals with strong multipoint correlations. In the cat primary visual cortex we compare responses to natural inputs with those to noise inputs matched for luminance and contrast. We find that neural filters adaptively change with the input ensemble so as to increase the information carried by the neural response about the filtered stimulus. Adaptation affects the spatial frequency composition of the filter, enhancing sensitivity to under-represented frequencies in agreement with optimal encoding arguments. Adaptation occurs over 40 s to many minutes, longer than most previously reported forms of adaptation.Comment: 20 pages, 11 figures, includes supplementary informatio

Temporal sampling, resetting, and adaptation orchestrate gradient sensing in sperm

© The Author(s), 2012. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Journal of Cell Biology 198 (2012): 1075-1091, doi:10.1083/jcb.201204024.Sperm, navigating in a chemical gradient, are exposed to a periodic stream of chemoattractant molecules. The periodic stimulation entrains Ca2+ oscillations that control looping steering responses. It is not known how sperm sample chemoattractant molecules during periodic stimulation and adjust their sensitivity. We report that sea urchin sperm sampled molecules for 0.2–0.6 s before a Ca2+ response was produced. Additional molecules delivered during a Ca2+ response reset the cell by causing a pronounced Ca2+ drop that terminated the response; this reset was followed by a new Ca2+ rise. After stimulation, sperm adapted their sensitivity following the Weber–Fechner law. Taking into account the single-molecule sensitivity, we estimate that sperm can register a minimal gradient of 0.8 fM/µm and be attracted from as far away as 4.7 mm. Many microorganisms sense stimulus gradients along periodic paths to translate a spatial distribution of the stimulus into a temporal pattern of the cell response. Orchestration of temporal sampling, resetting, and adaptation might control gradient sensing in such organisms as well.This work was supported by the German Research Foundation and the Fonds der Chemischen Industrie.2013-03-1

Electroretinogram Analysis of the Visual Response in Zebrafish Larvae

The electroretinogram (ERG) is a noninvasive electrophysiological method for determining retinal function. Through the placement of an electrode on the surface of the cornea, electrical activity generated in response to light can be measured and used to assess the activity of retinal cells in vivo. This manuscript describes the use of the ERG to measure visual function in zebrafish. Zebrafish have long been utilized as a model for vertebrate development due to the ease of gene suppression by morpholino oligonucleotides and pharmacological manipulation. At 5-10 dpf, only cones are functional in the larval retina. Therefore, the zebrafish, unlike other animals, is a powerful model system for the study of cone visual function in vivo. This protocol uses standard anesthesia, micromanipulation and stereomicroscopy protocols that are common in laboratories that perform zebrafish research. The outlined methods make use of standard electrophysiology equipment and a low light camera to guide the placement of the recording microelectrode onto the larval cornea. Finally, we demonstrate how a commercially available ERG stimulator/recorder originally designed for use with mice can easily be adapted for use with zebrafish. ERG of larval zebrafish provides an excellent method of assaying cone visual function in animals that have been modified by morpholino oligonucleotide injection as well as newer genome engineering techniques such as Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9, all of which have greatly increased the efficiency and efficacy of gene targeting in zebrafish. In addition, we take advantage of the ability of pharmacological agents to penetrate zebrafish larvae to evaluate the molecular components that contribute to the photoresponse. This protocol outlines a setup that can be modified and used by researchers with various experimental goals

Sulfhydryl Binding Reagents Increase the Conductivity of the Light-Sensitive Channel and Inhibit Phototransduction in Retinal Rods

The mechanisms by which sulfhydryl (SH-) binding reagents modulate the light-sensitive conductance of retinal rods were investigated by current recording from single rods, by patch clamp recording from the plasma membrane of the rod outer segment (ROS), and by biochemical study of their effects on the light-induced hydrolysis of cyclic GMP. The electrophysiology, as well as measurements of the reagents' ability to traverse the ROS plasma membrane, was done on amphibian (Rona and Ambystoma) rods, and the biochemistry on bovine rods. The main SH-reagents used were N-ethyl-maleimide (NEM) and iodoacetamide (IAA). Both transiently increased rod current, but part of the large current could not be turned off by light. After a few minutes' exposure, NEM, but not IAA, caused a continuous decay of the rod's light sensitivity. In patch-clamp recordings from the ROS plasma membrane, the reagents increased conductivity both in the presence and absence of cGMP, consistent with the observation that the drug-induced current increase in intact rods involved both light-sensitive and light-insensitive components. In vitro, NEM was found to be a powerful inhibitor of cGMP hydrolysis, which can explain the gradual loss of light sensitivity in the rod and could initially contribute to the increased dark current via elevated cGMP levels. Thus, SH-reagents act both by modifying the light-sensitive channel and by inhibiting phototransduction inside the rod

Speed, adaptation, and stability of the response to light in cone photoreceptors: The functional role of Ca-dependent modulation of ligand sensitivity in cGMP-gated ion channels

The response of cone photoreceptors to light is stable and reproducible because of the exceptional regulation of the cascade of enzymatic reactions that link visual pigment (VP) excitation to the gating of cyclic GMP (cGMP)-gated ion channels (cyclic nucleotide–gated [CNG]) in the outer segment plasma membrane. Regulation is achieved in part through negative feedback control of some of these reactions by cytoplasmic free Ca2+. As part of the control process, Ca2+ regulates the phosphorylation of excited VP, the activity of guanylate cyclase, and the ligand sensitivity of the CNG ion channels. We measured photocurrents elicited by stimuli in the form of flashes, steps, and flashes superimposed on steps in voltage-clamped single bass cones isolated from striped bass retina. We also developed a computational model that comprises all the known molecular events of cone phototransduction, including all Ca-dependent controls. Constrained by available experimental data in bass cones and cone transduction biochemistry, we achieved an excellent match between experimental photocurrents and those simulated by the model. We used the model to explore the physiological role of CNG ion channel modulation. Control of CNG channel activity by both cGMP and Ca2+ causes the time course of the light-dependent currents to be faster than if only cGMP controlled their activity. Channel modulation also plays a critical role in the regulation of the light sensitivity and light adaptation of the cone photoresponse. In the absence of ion channel modulation, cone photocurrents would be unstable, oscillating during and at the offset of light stimuli

An online spike detection and spike classification algorithm capable of instantaneous resolution of overlapping spikes

For the analysis of neuronal cooperativity, simultaneously recorded extracellular signals from neighboring neurons need to be sorted reliably by a spike sorting method. Many algorithms have been developed to this end, however, to date, none of them manages to fulfill a set of demanding requirements. In particular, it is desirable to have an algorithm that operates online, detects and classifies overlapping spikes in real time, and that adapts to non-stationary data. Here, we present a combined spike detection and classification algorithm, which explicitly addresses these issues. Our approach makes use of linear filters to find a new representation of the data and to optimally enhance the signal-to-noise ratio. We introduce a method called “Deconfusion” which de-correlates the filter outputs and provides source separation. Finally, a set of well-defined thresholds is applied and leads to simultaneous spike detection and spike classification. By incorporating a direct feedback, the algorithm adapts to non-stationary data and is, therefore, well suited for acute recordings. We evaluate our method on simulated and experimental data, including simultaneous intra/extra-cellular recordings made in slices of a rat cortex and recordings from the prefrontal cortex of awake behaving macaques. We compare the results to existing spike detection as well as spike sorting methods. We conclude that our algorithm meets all of the mentioned requirements and outperforms other methods under realistic signal-to-noise ratios and in the presence of overlapping spikes