Investigating the Ca2+-dependent and Ca2+-independent mechanisms for mammalian cone light adaptation

Abstract Vision is mediated by two types of photoreceptors: rods, enabling vision in dim light; and cones, which function in bright light. Despite many similarities in the components of their respective phototransduction cascades, rods and cones have distinct sensitivity, response kinetics, and adaptation capacity. Cones are less sensitive and have faster responses than rods. In addition, cones can function over a wide range of light conditions whereas rods saturate in moderately bright light. Calcium plays an important role in regulating phototransduction and light adaptation of rods and cones. Notably, the two dominant Ca2+-feedbacks in rods and cones are driven by the identical calcium-binding proteins: guanylyl cyclase activating proteins 1 and 2 (GCAPs), which upregulate the production of cGMP; and recoverin, which regulates the inactivation of visual pigment. Thus, the mechanisms producing the difference in adaptation capacity between rods and cones have remained poorly understood. Using GCAPs/recoverin-deficient mice, we show that mammalian cones possess another Ca2+-dependent mechanism promoting light adaptation. Surprisingly, we also find that, unlike in mouse rods, a unique Ca2+-independent mechanism contributes to cone light adaptation. Our findings point to two novel adaptation mechanisms in mouse cones that likely contribute to the great adaptation capacity of cones over rods

Calcium Sets the Physiological Value of the Dominant Time Constant of Saturated Mouse Rod Photoresponse Recovery

Background: The rate-limiting step that determines the dominant time constant (tD) of mammalian rod photoresponse recovery is the deactivation of the active phosphodiesterase (PDE6). Physiologically relevant Ca 2+-dependent mechanisms that would affect the PDE inactivation have not been identified. However, recently it has been shown that tD is modulated by background light in mouse rods. Methodology/Principal Findings: We used ex vivo ERG technique to record pharmacologically isolated photoreceptor responses (fast PIII component). We show a novel static effect of calcium on mouse rod phototransduction: Ca 2+ shortens the dominant time constant (tD) of saturated photoresponse recovery, i.e., when extracellular free Ca 2+ is decreased from 1mMto,25 nM, the tD is reversibly increased,1.5–2-fold. Conclusions: We conclude that the increase in tD during low Ca 2+ treatment is not due to increased [cGMP], increased [Na +] or decreased [ATP] in rod outer segment (ROS). Also it cannot be due to protein translocation mechanisms. We suggest that aCa 2+-dependent mechanism controls the life time of active PDE

The Na+/Ca2+, K+ exchanger 2 modulates mammalian cone phototransduction

Calcium ions (Ca(2+)) modulate the phototransduction cascade of vertebrate cone photoreceptors to tune gain, inactivation, and light adaptation. In darkness, the continuous current entering the cone outer segment through cGMP-gated (CNG) channels is carried in part by Ca(2+), which is then extruded back to the extracellular space. The mechanism of Ca(2+) extrusion from mammalian cones is not understood. The dominant view has been that the cone-specific isoform of the Na(+)/Ca(2+), K(+) exchanger, NCKX2, is responsible for removing Ca(2+) from their outer segments. However, indirect evaluation of cone function in NCKX2-deficient (Nckx2(−/−)) mice by electroretinogram recordings revealed normal photopic b-wave responses. This unexpected result suggested that NCKX2 may not be involved in the Ca(2+) homeostasis of mammalian cones. To address this controversy, we examined the expression of NCKX2 in mouse cones and performed transretinal recordings from Nckx2(−/−) mice to determine the effect of NCKX2 deletion on cone function directly. We found that Nckx2(−/−) cones exhibit compromised phototransduction inactivation, slower response recovery and delayed background adaptation. We conclude that NCKX2 is required for the maintenance of efficient Ca(2+) extrusion from mouse cones. However, surprisingly, Nckx2(−/−) cones adapted normally in steady background light, indicating the existence of additional Ca(2+)-extruding mechanisms in mammalian cones

Guanylate cyclase–activating protein 2 contributes to phototransduction and light adaptation in mouse cone photoreceptors

Light adaptation of photoreceptor cells is mediated by Ca2+-dependent mechanisms. In darkness, Ca2+ influx through cGMP-gated channels into the outer segment of photoreceptors is balanced by Ca2+ extrusion via Na+/Ca2+, K+ exchangers (NCKXs). Light activates a G protein signaling cascade, which closes cGMP-gated channels and decreases Ca2+ levels in photoreceptor outer segment because of continuing Ca2+ extrusion by NCKXs. Guanylate cyclase-activating proteins (GCAPs) then up-regulate cGMP synthesis by activating retinal membrane guanylate cyclases (RetGCs) in low Ca2+ This activation of RetGC accelerates photoresponse recovery and critically contributes to light adaptation of the nighttime rod and daytime cone photoreceptors. In mouse rod photoreceptors, GCAP1 and GCAP2 both contribute to the Ca2+-feedback mechanism. In contrast, only GCAP1 appears to modulate RetGC activity in mouse cones because evidence of GCAP2 expression in cones is lacking. Surprisingly, we found that GCAP2 is expressed in cones and can regulate light sensitivity and response kinetics as well as light adaptation of GCAP1-deficient mouse cones. Furthermore, we show that GCAP2 promotes cGMP synthesis and cGMP-gated channel opening in mouse cones exposed to low Ca2+ Our biochemical model and experiments indicate that GCAP2 significantly contributes to the activation of RetGC1 at low Ca2+ when GCAP1 is not present. Of note, in WT mouse cones, GCAP1 dominates the regulation of cGMP synthesis. We conclude that, under normal physiological conditions, GCAP1 dominates the regulation of cGMP synthesis in mouse cones, but if its function becomes compromised, GCAP2 contributes to the regulation of phototransduction and light adaptation of cones

Visual Neuroscience Flash Responses of Mouse Rod Photoreceptors in the Isolated Retina and Corneal Electroretinogram: Comparison of Gain and Kinetics

PURPOSE. To examine the amplification and kinetics of murine rod photoresponses by recording ERG flash responses in vivo and ex vivo from the same retina. We also aimed to evaluate the two available methods for isolating the rod signal from the ERG flash response, that is, pharmacology and paired flash method on the isolated retina. METHODS. Dark-adapted ERG responses to full-field flashes of green light were recorded from anesthetized (ketamine/ xylazine) C57BL/6N mice. ERG flash responses to homogenous light stimuli arriving from the photoreceptor side were then recorded transretinally from the same retinas, isolated and perfused with Ringer's or Ames' solution at 378C. The responses were analyzed to determine the a-wave kinetics as well as the estimated flash sensitivity and kinetics of the full rod responses derived with the paired flash protocol. The analysis was complemented with pharmacologic blockade of glutamatergic transmission in the isolated retina. RESULTS. The a-waves were of comparable size, sensitivity and kinetics in vivo and in the isolated retina, but the onset of the b-wave was delayed in the isolated retina. The Lamb-Pugh activation constants determined for the a-waves were similar in both preparations. The kinetics of the derived photoreceptor responses were similar in both conditions, although the responses were consistently slightly slower ex vivo. This was not explicable as a direct effect of ketamine or xylazine on the photoreceptors or as their indirect effect through hyperglycemia, as tested on the isolated retina. CONCLUSIONS. Through comparison to the corneal ERG, the transretinal ERG is a valuable tool for assaying the physiologic state of isolated retinal tissue. The rod photoreceptor responses of the intact isolated retina correspond well to those recorded in vivo. The origin of their faster kinetics compared to single cell recordings remains to be determined. (Invest Ophthalmol Vis Sci. 2012;53:5653-5664

Mitochondrial Calcium Uniporter (MCU) defciency reveals an alternate path for ­Ca2+ uptake in photoreceptor mitochondria

Rods and cones use intracellular Ca2+ to regulate many functions, including phototransduction and neurotransmission. The Mitochondrial Calcium Uniporter (MCU) complex is thought to be the primary pathway for Ca2+ entry into mitochondria in eukaryotes. We investigate the hypothesis that mitochondrial Ca2+ uptake via MCU influences phototransduction and energy metabolism in photoreceptors using a mcu-/- zebrafish and a rod photoreceptor-specific Mcu-/- mouse. Using genetically encoded Ca2+ sensors to directly examine Ca2+ uptake in zebrafish cone mitochondria, we found that loss of MCU reduces but does not eliminate mitochondrial Ca2+ uptake. Loss of MCU does not lead to photoreceptor degeneration, mildly affects mitochondrial metabolism, and does not alter physiological responses to light, even in the absence of the Na+/Ca2+, K+ exchanger. Our results reveal that MCU is dispensable for vertebrate photoreceptor function, consistent with its low expression and the presence of an alternative pathway for Ca2+ uptake into photoreceptor mitochondria

The Na+/Ca2+, K+ exchanger 2 modulates mammalian cone phototransduction

Calcium ions (Ca2+) modulate the phototransduction cascade of vertebrate cone photoreceptors to tune gain, inactivation, and light adaptation. In darkness, the continuous current entering the cone outer segment through cGMP-gated (CNG) channels is carried in part by Ca2+, which is then extruded back to the extracellular space. The mechanism of Ca2+ extrusion from mammalian cones is not understood. The dominant view has been that the cone-specific isoform of the Na+/Ca2+, K+ exchanger, NCKX2, is responsible for removing Ca2+ from their outer segments. However, indirect evaluation of cone function in NCKX2-deficient (Nckx2−/−) mice by electroretinogram recordings revealed normal photopic b-wave responses. This unexpected result suggested that NCKX2 may not be involved in the Ca2+ homeostasis of mammalian cones. To address this controversy, we examined the expression of NCKX2 in mouse cones and performed transretinal recordings from Nckx2−/− mice to determine the effect of NCKX2 deletion on cone function directly. We found that Nckx2−/− cones exhibit compromised phototransduction inactivation, slower response recovery and delayed background adaptation. We conclude that NCKX2 is required for the maintenance of efficient Ca2+ extrusion from mouse cones. However, surprisingly, Nckx2−/− cones adapted normally in steady background light, indicating the existence of additional Ca2+-extruding mechanisms in mammalian cones

Ionic mechanisms in mouse rod photoreceptor signaling

Photoreceptor cells are an example of biological transducer devices: they transform photon energy into an electrical signal and transmit it to higher-order neurons. Vertebrate photoreceptor cells can be categorized into two classes, rods and cones. The rod photoreceptors are extremely sensitive to light, whereas cones are faster than rods and can function under bright ambient illumination. The rod photoreceptor cell is a convenient model for studying modulation of physiological transduction and transmission processes because 1) the rod's natural input signal, light, can be applied quantitatively and 2) the absorption of only one or a few photons by the visual pigment molecules in the cell's outer segment is transformed into a measurable change of the rod's membrane potential (Vm). The gain of the photon-to-Vm conversion in rods is rapidly (in a fraction of a second) modulated by several ionic feedback mechanisms. The mechanisms involved in rod signal generation and feedback signaling were investigated in the present work by recording rods' electrical responses to light stimuli from intact mouse retinas with transretinal electroretinogram (ex vivo ERG). Several negative feedback mechanisms that accelerate a rod's response recovery after light stimuli rely on the light-induced decline in the calcium ion (Ca2+) concentration in the rod outer segment. Further, some voltage- and Ca2+ -dependent mechanisms in the rod inner segment plasma membrane modulate the gain of the photon-to-Vm conversion. In this thesis the specificity of the known Ca2+ signaling mechanisms, and Ca2+ dependency of the reaction that rate-limits the rod's recovery after bright stimuli were investigated. It was found that the transition metal ion Co2+ can mediate the known Ca2+ dependent negative feedback mechanisms in the rod outer segment, and that a certain minimum amount of Ca2+ is necessary in setting the physiological value of the speed of the rate-limiting recovery reaction. The role of the inner segment ionic channels in generating the rod ERG response was also elucidated. It was shown that the hyperpolarization activated (h) channels in the rod inner segment participate in the generation of a fast transient component that is evident in the rod ERG response to bright flashes. Instead, voltage-dependent Ca2+ channels or Ca2+ -activated potassium and chloride currents did not contribute to that component. Additionally, modulation of the direct electrical transmission between rods and cones was studied. The present work suggests that the electrical connection between rods and cones can be closed by light in the mouse retina

Hiiren eristetyn verkkokalvon ERG:n nopean PIII-komponentin tutkimus

Elektroretinogrammimenetelmässä (ERG) mitataan verkkokalvon yli vallitsevaa jännitettä, joka muodostuu solunulkoisten radiaalisten virtojen aiheuttamista jännitehäviöistä. ERG-signaali syntyy, kun solunulkoiset radiaaliset virrat muuttuvat verkkokalvon altistuessa valolle. Signaali sisältää useita komponentteja, joiden solu- ja molekyylitason mekanismien tunteminen on tärkeää, jotta ERG-menetelmää voidaan käyttää luotettavasti tutkimuksessa ja kliinisessä työssä. Verkkokalvo koostuu useasta hermosolukerroksesta ja niitä yhdistävistä synaptisista kerroksista. Varsinaiset valon absorboimiseen erikoistuneet hermosolut (ensimmäisen kertaluvun neuronit), näköaistinsolut, sijaitsevat verkkokalvon distaalisella pinnalla. Näköaistinsolut voidaan jakaa rakenteellisten ja toiminnallisten ominaisuuksien perusteella kahteen ryhmään: sauvasoluihin ja tappisoluihin. Sauvasolut ovat erikoistuneet näkemään pimeässä (skotooppinen näkö) ja ovat huomattavasti herkempiä kuin tappisolut. Arden (1976) kuvasi ensimmäisen kerran rotan eristetyn verkkokalvon ERG-signaalista erittäin nopean negatiivisen aallon, jonka hän nimesi 'nenäkomponentiksi'. Nenäkomponentti (n-aalto) oli nähtävissä selkeimmin, kun Arden käytti kirkkaita sauvasolut saturoivia valostimuluksia. Tämän diplomityön tavoitteena oli selvittää n-aallon molekyylitason syntymekanismeja. Tässä diplomityössä n-aaltoa tutkittiin hiiren (Mus musculus) eristetyn verkkokalvon ERG-tekniikalla. Valostimuluksena käytettiin vihreää (543,5 nm, pulssin pituus 20 ms) laservaloa ja verkkokalvon lämpötila oli mittausten aikana 25 Celsius-astetta. Verkkokalvoa perfusoitiin fysiologisella suolaliuoksella näköaistinsolujen puolelta. n-komponentin synnyttävän virran molekyylitason lähde- ja nielumekanismeja pyrittiin selvittämään moduloimalla tunnettuja mahdollisia mekanismeja farmakologisesti. Diplomityössä tehtyjen kokeiden perusteella n-aalto syntyy todennäköisesti näköaistinsolukerroksessa ja nimenomaan sauvasolujen sähköisen aktiivisuuden seurauksena: (i) Kaikissa kokeissa kemiallinen signaalivälitys korkeamman kertaluvun neuroneihin oli estetty, mikä ei poistanut n-aaltoa. (ii) Kaksoisstimulusmenetelmässä tappisignaalissa ei nähty n-aaltoa. (iii) Koetulokset eivät myöskään tue aukkoliitosten tai kapasitiivisen virran osuutta n-aallon syntymekanismissa. n-aallon nieluna toimivat erittäin todennäköisesti sauvasolujen sisäjäsenessä sijaitsevat hyperpolarisaation seurauksena aktivoituvat h-kanavat. Tätä päätelmää tukevat useat koetulokset: (i) h-kanavia salpaava cesium poisti n-aallon lähes kokonaan. (ii) h-kanavien kinetiikkaa hidastava ja h-virtaa pienentävä niflumihappo hidasti ja pienensi n-aaltoa. (iii) Solukalvoa depolarisoivat farmakologiset manipuloinnit pienensivät n-aaltoa. Tässä diplomityössä ei löydetty n-aallon lähdettä. Koetulosten perusteella näköaistinsolujen synapsin alueen L-tyypin kalsiumvirrat tai kalsiumohjatut kloridivirrat eivät liity n-aallon syntymekanismiin