Sensitivity and kinetics of signal transmission at the first visual synapse differentially impact visually-guided behavior

In the retina, synaptic transmission between photoreceptors and downstream ON-bipolar neurons (ON-BCs) is mediated by a GPCR pathway, which plays an essential role in vision. However, the mechanisms that control signal transmission at this synapse and its relevance to behavior remain poorly understood. In this study we used a genetic system to titrate the rate of GPCR signaling in ON-BC dendrites by varying the concentration of key RGS proteins and measuring the impact on transmission of signal between photoreceptors and ON-BC neurons using electroretinography and single cell recordings. We found that sensitivity, onset timing, and the maximal amplitude of light-evoked responses in rod- and cone-driven ON-BCs are determined by different RGS concentrations. We further show that changes in RGS concentration differentially impact visually guided-behavior mediated by rod and cone ON pathways. These findings illustrate that neuronal circuit properties can be modulated by adjusting parameters of GPCR-based neurotransmission at individual synapses

Exchange of Cone for Rod Phosphodiesterase 6 Catalytic Subunits in Rod Photoreceptors Mimics in Part Features of Light Adaptation

Despite the expression of homologous phototransduction components, the molecular basis for differences in light-evoked responses between rod and cone photoreceptors remains unclear. We examined the role of cGMP phosphodiesterase (PDE6) in this difference by expressing cone PDE6 (PDE6C) in rd1/rd1 rods lacking rod PDE6 (PDE6AB) using transgenic mice. The expression of PDE6C rescues retinal degeneration observed in rd1/rd1 rods. Double-transgenic rods (PDE6C++) were compared with rd1/+ rods based on similar PDE6 expression. PDE6C increased the basal PDE activity and speeded the rate-limiting step for phototransduction deactivation, causing rod photoresponses to appear light adapted, with reduced dark current and sensitivity and faster response kinetics. When PDE6C++ and rd1/+ rods were exposed to similar background light, rd1/+ rods displayed greater desensitization. These results indicate an increased spontaneous activity and faster deactivation of PDE6C compared with PDE6AB in darkness, but that background light increases steady PDE6C activity to a lesser extent. In addition to accelerating the recovery of the photoresponse, faster PDE6C deactivation may blunt the rise in background-induced steady PDE6C activity. Therefore, higher basal PDE6C activity and faster deactivation together partially account for faster and less sensitive cone photoresponses in darkness, whereas a reduced rise of steady PDE6C activity in background light may allow cones to avoid saturation. Significance Statement Cones are the primary photoreceptors responsible for most of our visual experience. Cone light responses are less sensitive and display speeded responses compared with rods. Despite the fact that rods and cones use a G-protein signaling cascade with similar organization, the mechanistic basis for these differences remains unclear. Here, we examined the role of distinct isoforms of PDE6, the effector enzyme in phototransduction, in these differences. We developed a transgenic mouse model that expresses cone PDE6 in rods and show that the cone PDE6 isoform is partially responsible for the difference in sensitivity and response kinetics between rods and cones

Apoptosis Regulates ipRGC Spacing Necessary for Rods and Cones to Drive Circadian Photoentrainment

SummaryThe retina consists of ordered arrays of individual types of neurons for processing vision. Here, we show that such order is necessary for intrinsically photosensitive retinal ganglion cells (ipRGCs) to function as irradiance detectors. We found that during development, ipRGCs undergo proximity-dependent Bax-mediated apoptosis. Bax mutant mice exhibit disrupted ipRGC spacing and dendritic stratification with an increase in abnormally localized synapses. ipRGCs are the sole conduit for light input to circadian photoentrainment, and either their melanopsin-based photosensitivity or ability to relay rod/cone input is sufficient for circadian photoentrainment. Remarkably, the disrupted ipRGC spacing does not affect melanopsin-based circadian photoentrainment but severely impairs rod/cone-driven photoentrainment. We demonstrate reduced rod/cone-driven cFos activation and electrophysiological responses in ipRGCs, suggesting that impaired synaptic input to ipRGCs underlies the photoentrainment deficits. Thus, for irradiance detection, developmental apoptosis is necessary for the spacing and connectivity of ipRGCs that underlie their functioning within a neural network

Coordinated control of sensitivity by two splice variants of Gαo in retinal ON bipolar cells

The high sensitivity of scotopic vision depends on the efficient retinal processing of single photon responses generated by individual rod photoreceptors. At the first synapse in the mammalian retina, rod outputs are pooled by a rod “ON” bipolar cell, which uses a G-protein signaling cascade to enhance the fidelity of the single photon response under conditions where few rods absorb light. Here we show in mouse rod bipolar cells that both splice variants of the Go α subunit, Gαo1 and Gαo2, mediate light responses under the control of mGluR6 receptors, and their coordinated action is critical for maximizing sensitivity. We found that the light response of rod bipolar cells was primarily mediated by Gαo1, but the loss of Gαo2 caused a reduction in the light sensitivity. This reduced sensitivity was not attributable to the reduction in the total number of Go α subunits, or the altered balance of expression levels between the two splice variants. These results indicate that Gαo1 and Gαo2 both mediate a depolarizing light response in rod bipolar cells without occluding each other’s actions, suggesting they might act independently on a common effector. Thus, Gαo2 plays a role in improving the sensitivity of rod bipolar cells through its action with Gαo1. The coordinated action of two splice variants of a single Gα may represent a novel mechanism for the fine control of G-protein activity

Spectral Tuning and Adaptation to Different Light Environments of Mysid Visual Pigments

In the present thesis, questions of spectral tuning, the relation of spectral and thermal properties of visual pigments, and evolutionary adaptation to different light environments were addressed using a group of small crustaceans of the genus Mysis as a model. The study was based on microspectrophotometric measurements of visual pigment absorbance spectra, electrophysiological measurements of spectral sensitivities of dark-adapted eyes, and sequencing of the opsin gene retrieved through PCR. The spectral properties were related to the spectral transmission of the respective light environments, as well as to the phylogentic histories of the species. The photoactivation energy (Ea) was estimated from temperature effects on spectral sensitivity in the long-wavelength range, and calculations were made for optimal quantum catch and optimal signal-to-noise ratio in the different light environments. The opsin amino acid sequences of spectrally characterized individuals were compared to find candidate residues for spectral tuning. The general purpose was to clarify to what extent and on what time scale adaptive evolution has driven the functional properties of (mysid) visual pigments towards optimal performance in different light environments. An ultimate goal was to find the molecular mechanisms underlying the spectral tuning and to understand the balance between evolutionary adaptation and molecular constraints. The totally consistent segregation of absorption maxima (λmax) into (shorter-wavelength) marine and (longer-wavelength) freshwater populations suggests that truly adaptive evolution is involved in tuning the visual pigment for optimal performance, driven by selection for high absolute visual sensitivity. On the other hand, the similarity in λmax and opsin sequence between several populations of freshwater M. relicta in spectrally different lakes highlights the limits to adaptation set by evolutionary history and time. A strong inverse correlation between Ea and λmax was found among all visual pigments studied in these respects, including those of M. relicta and 10 species of vertebrate pigments, and this was used to infer thermal noise. The conceptual signal-to-noise ratios thus calculated for pigments with different λmax in the Baltic Sea and Lake Pääjärvi light environments supported the notion that spectral adaptation works towards maximizing the signal-to-noise ratio rather than quantum catch as such. Judged by the shape of absorbance spectra, the visual pigments of all populations of M. relicta and M. salemaai used exclusively the A2 chromophore (3, 4-dehydroretinal). A comparison of amino acid substitutions between M. relicta and M. salemaai indicated that mysid shrimps have a small number of readily available tuning sites to shift between a shorter - and a longer -wavelength opsin. However, phylogenetic history seems to have prevented marine M. relicta from converting back to the (presumably) ancestral opsin form, and thus the more recent reinvention of marine spectral sensitivity has been accomplished by some other novel mechanism, yet to be foundKaikki näköaistin käytettävissä oleva informaatio on peräisin fotoreseptorisoluista, joiden tehtävänä on aistia valoa. Fotoreseptorin näköpigmenttimolekyyli, rodopsiini, koostuu transmembraanireseptoriosasta (opsiini), sekä siihen sitoutuneesta valoherkästä A-vitamiinialdehydistä (kromofori). Eläimen näkö on suoraan riippuvainen näköpigmenttimolekyylien absorptiospektristä, eli siitä, mitä valon eri aallonpituuksia ne pystyvät absorboimaan. Jokaisella pigmentillä on tietty absorptiomaksimi, missä fotonin absorptiotodennäköisyys on korkein. Näköpigmenttimolekyylin pitäisikin pystyä absorboimaan mahdollisimman hyvin juuri sitä valon aallonpituusaluetta, jota eläimen ympäristössä on tarjolla. Samalla sen tulisi olla mahdollisimman stabiili, jotta kohinaa, eli (termistä) sisäistä satunnaisaktivoitumista, muodostuisi vähän ja signaali/kohina-suhde olisi mahdollisimman korkea. Väitöskirjassa tarkasteltiin näköpigmentin spektraaliherkkyyttä ja termisiä ominaisuuksia, niiden riippuvuutta toisistaan, sekä näköpigmentin sopeutumista eri valoympäristöihin. Tutkimuksessa mallina oli Mysis halkoisjalkais-äyriäisiä sekä murto- että sisävesistöistä, joissa nämä eläimet ovat sopeutuneet eri valoympäristöihin. Mysis-äyriäisiin kuuluu useita geneettisesti määriteltyjä lajeja, joiden syntyhistoria tunnetaan melko hyvin (mm jääkauden erottamat lajit). Tutkimuksen tavoitteena oli selvittää millä aikavälillä evolutiivinen sopeutuminen on optimoinut näköaistin toimintaa ja mitkä ovat ne selektiiviset paineet, jotka muokkaavat näköaistia. Väitöskirjatyössä selvitettiin myös molekyylitason mekanismeja, jotka säätelevät näköpigmentin toiminnallisia ominaisuuksia. Meri- ja makeavesi populaatioiden absorptiospektrit erosivat toisistaan kaikissa tutkituissa lajeissa. Tulokset osoittavat, että noin 9000 vuodessa evolutiivinen adaptaatio on muokannut valoaistin toimintaa valoympäristöön sopivaksi, jossa selektiivinen paine on ollut valoherkkyyden optimoinnissa. Laskennalliset arviot termisten ja spektraalisten ominaisuuksien riippuvuudesta toisistaan osoittivat, että Mysis näköpigmentin sopeutumisessa eri valoympäristöihin signaali/kohina suhdetta parannetaan minimoimalla sisäistä satunnaisaktiviteettiä, eikä niinkään fotonisaaliin, eli absorptiomaksimin maksimoinilla. Tietyissä makeanveden populaatioissa havaittiin kuitenkin yhtäläinen absorptiomaksimi eri valoympäristöissä, joten myös fylogeneettinen historia ja aika olivat asettaneet rajoituksia näköpigmentin sopeutumiseen. Makean- ja murtoveden Mysis-populaatioiden näköpigmenttien opsiinisekvenssien vertailu osoitti, että Mysis-lajeilla on vain tietty rajallinen määrä mahdollisuuksia muuttaa aminohapposekvenssiään suuntaan, joka tuottaisi kullekin populaatiolle sen valoympäristöön sopivimman näköpigmentin

Coordinated control of sensitivity by two splice variants of Gα(o) in retinal ON bipolar cells.

The high sensitivity of scotopic vision depends on the efficient retinal processing of single photon responses generated by individual rod photoreceptors. At the first synapse in the mammalian retina, rod outputs are pooled by a rod "ON" bipolar cell, which uses a G-protein signaling cascade to enhance the fidelity of the single photon response under conditions where few rods absorb light. Here we show in mouse rod bipolar cells that both splice variants of the G(o) α subunit, Gα(o1) and Gα(o2), mediate light responses under the control of mGluR6 receptors, and their coordinated action is critical for maximizing sensitivity. We found that the light response of rod bipolar cells was primarily mediated by Gα(o1), but the loss of Gα(o2) caused a reduction in the light sensitivity. This reduced sensitivity was not attributable to the reduction in the total number of G(o) α subunits, or the altered balance of expression levels between the two splice variants. These results indicate that Gα(o1) and Gα(o2) both mediate a depolarizing light response in rod bipolar cells without occluding each other's actions, suggesting they might act independently on a common effector. Thus, Gα(o2) plays a role in improving the sensitivity of rod bipolar cells through its action with Gα(o1). The coordinated action of two splice variants of a single Gα may represent a novel mechanism for the fine control of G-protein activity

Exchange of Cone for Rod Phosphodiesterase 6 Catalytic Subunits in Rod Photoreceptors Mimics in Part Features of Light Adaptation.

UnlabelledDespite the expression of homologous phototransduction components, the molecular basis for differences in light-evoked responses between rod and cone photoreceptors remains unclear. We examined the role of cGMP phosphodiesterase (PDE6) in this difference by expressing cone PDE6 (PDE6C) in rd1/rd1 rods lacking rod PDE6 (PDE6AB) using transgenic mice. The expression of PDE6C rescues retinal degeneration observed in rd1/rd1 rods. Double-transgenic rods (PDE6C++) were compared with rd1/+ rods based on similar PDE6 expression. PDE6C increased the basal PDE activity and speeded the rate-limiting step for phototransduction deactivation, causing rod photoresponses to appear light adapted, with reduced dark current and sensitivity and faster response kinetics. When PDE6C++ and rd1/+ rods were exposed to similar background light, rd1/+ rods displayed greater desensitization. These results indicate an increased spontaneous activity and faster deactivation of PDE6C compared with PDE6AB in darkness, but that background light increases steady PDE6C activity to a lesser extent. In addition to accelerating the recovery of the photoresponse, faster PDE6C deactivation may blunt the rise in background-induced steady PDE6C activity. Therefore, higher basal PDE6C activity and faster deactivation together partially account for faster and less sensitive cone photoresponses in darkness, whereas a reduced rise of steady PDE6C activity in background light may allow cones to avoid saturation.Significance statementCones are the primary photoreceptors responsible for most of our visual experience. Cone light responses are less sensitive and display speeded responses compared with rods. Despite the fact that rods and cones use a G-protein signaling cascade with similar organization, the mechanistic basis for these differences remains unclear. Here, we examined the role of distinct isoforms of PDE6, the effector enzyme in phototransduction, in these differences. We developed a transgenic mouse model that expresses cone PDE6 in rods and show that the cone PDE6 isoform is partially responsible for the difference in sensitivity and response kinetics between rods and cones

Detection of single photons by toad and mouse rods.

Amphibian and mammalian rods can both detect single photons of light even though they differ greatly in physical dimensions, mammalian rods being much smaller in diameter than amphibian rods. To understand the changes in physiology and biochemistry required by such large differences in outer segment geometry, we developed a computational approach, taking into account the spatial organization of the outer segment divided into compartments, together with molecular dynamics simulations of the signaling cascade. We generated simulations of the single-photon response together with intrinsic background fluctuations in toad and mouse rods. Combining this computational approach with electrophysiological data from mouse rods, we determined key biochemical parameters. On average around one phosphodiesterase (PDE) molecule is spontaneously active per mouse compartment, similar to the value for toad, which is unexpected due to the much smaller diameter in mouse. A larger number of spontaneously active PDEs decreases dark noise, thereby improving detection of single photons; it also increases cGMP turnover, which accelerates the decay of the light response. These constraints explain the higher PDE density in mammalian compared with amphibian rods that compensates for the much smaller diameter of mammalian disks. We further find that the rate of cGMP hydrolysis by light-activated PDE is diffusion limited, which is not the case for spontaneously activated PDE. As a consequence, in the small outer segment of a mouse rod only a few activated PDEs are sufficient to generate a signal that overcomes noise, which permits a shorter lifetime of activated rhodopsin and greater temporal resolution

Detection of single photons by toad and mouse rods.

Amphibian and mammalian rods can both detect single photons of light even though they differ greatly in physical dimensions, mammalian rods being much smaller in diameter than amphibian rods. To understand the changes in physiology and biochemistry required by such large differences in outer segment geometry, we developed a computational approach, taking into account the spatial organization of the outer segment divided into compartments, together with molecular dynamics simulations of the signaling cascade. We generated simulations of the single-photon response together with intrinsic background fluctuations in toad and mouse rods. Combining this computational approach with electrophysiological data from mouse rods, we determined key biochemical parameters. On average around one phosphodiesterase (PDE) molecule is spontaneously active per mouse compartment, similar to the value for toad, which is unexpected due to the much smaller diameter in mouse. A larger number of spontaneously active PDEs decreases dark noise, thereby improving detection of single photons; it also increases cGMP turnover, which accelerates the decay of the light response. These constraints explain the higher PDE density in mammalian compared with amphibian rods that compensates for the much smaller diameter of mammalian disks. We further find that the rate of cGMP hydrolysis by light-activated PDE is diffusion limited, which is not the case for spontaneously activated PDE. As a consequence, in the small outer segment of a mouse rod only a few activated PDEs are sufficient to generate a signal that overcomes noise, which permits a shorter lifetime of activated rhodopsin and greater temporal resolution