King-Wai Yau - US grants

The long-term objective of the proposed research is to understand the cellular mechanisms underlying the detection and signaling of light stimuli by vertebrate retinal photoreceptors. The specific aims are to study: 1) the nature of the transduction stages between light absorption by visual pigment and ultimate generation of an electrical signal, 2) the involvement of sodium, calcium and other ions in transduction, 3) the involvement of cyclic nucleotides and related biochemical substrates in transduction, 4) the characteristics of the light-sensitive conductance, which gives rise to the ultimate electrical light response to be transmitted to higher neurons, 5) any possible fundamental differences between rods and cones in their transduction mechanisms, and 6) spontaneous excitation of visual pigments in darkness. Knowledge of visual transduction is important for understanding various disease states affecting photoreceptors and for devising remedies for them. The methodology will involve mainly electrophysiological recording, consisting in extracellularly recording membrane current from single rods and cones using a suction pipet, together with intracellular recording. The extracellular current recording method has unusual stability and high measurement resolution, and is very suitable for studying visual transduction, especially if employed in conjunction with pharmacology.

DESCRIPTION (provided by applicant): The long-term objective of the proposed research is to understand in detail the phototransduction mechanisms in retinal rods and cones and in other vertebrate photoreceptors. The specific aims are: 1) to understand why cones are much less sensitive to light than rods, and have response kinetics several-fold faster. We shall focus on cone pigments, particularly their tendency to dissociate into the apoprotein and 11-cis retinal (without isomerization), and its tendency to isomerize thermally at a high rate to the active state. 2) to continue study of phototransduction in the lizard parietal-eye photoreceptor. In particular, we would like to understand the chromatic antagonism in this cell, in which green light activates a cGMP-activated channel, while blue light closes the channel, acting through opposite effects on the cGMP-phosphodiesterase. We propose to characterize the detailed physiology and the biochemical/molecular aspects of this antagonism. The hope is to use this unusual system to shed new light on the G-protein control of the cGMP-phosphodiesterase. 3) to study the assembly of the rod and cone cyclic nucleotide-activated channels. In particular, we shall examine in detail a domain on the alpha-subunits of these channels identified by us that appears to be involved in channel assembly. We shall also examine whether this domain has to do with the selective assembly of the alpha- and beta-subunits of these channels into hetero-oligomers in rods and cones instead of perfectly functional alpha-homomeric channels. The methodology involves a combination of electrophysiology, molecular biology and biochemistry.

[unreadable] DESCRIPTION (provided by applicant): Besides the rod/cone pathway, a melanopsin-associated photoreception pathway is now known to exist in the retina. This pathway involves melanopsin-expressing retinal ganglion cells (mRGCs), a small subset of RGCs that are intrinsically photosensitive but also receive synaptic inputs from the rod/cone pathway. Unlike the non-mRGCs, which project predominantly to image-forming visual centers in the brain (the dorsal lateral geniculate nucleus and the superior colliculus), the mRGCs project primarily to non-image-forming (accessory) visual centers such as the suprachiasmatic nucleus and the intergeniculate leaflet (for circadian photoentrainment) as well as the olivary pretectal nucleus (for pupillary light reflex). Thus, there appears to be a functional segregation between non-mRGCs and mRGCs, with the mRGCs not only contributing to non-image-forming photoreception but also constituting the major conduit for all non-image-associated photic information to the brain. Besides the rod/cone system and the melanopsin system, there appears to be no other photoreception system in the eye that signal to the brain. The long-term objective of this proposal is to understand the light responses and the signaling by mRGCs in great detail. Aim 1 is to investigate the general physiological and biophysical properties of the light responses of mRGCs in detail. Aim 2 is to investigate light adaptation by mRGCs. Aim 3 is to investigate the conversion of the receptor potential into action potentials in the mRGCs, and the roles played by the various voltage-gated ion channels. Aim 4 is to understand the phototransduction mechanism in mRGCs and to identify the various proteins involved in this process. Both electrophysiology and mouse genetics will be used for study. These experiments will yield important information not only relevant to the basic understanding of this newly discovered melanopsin system, but also to disease states affecting light detection in the eye. PUBLIC HEALTH RELEVANCE: The studies proposed in this application will enhance our understanding of the newly discovered melanopsin-associated photoreception system in the retina. Any new information derived from these studies will also be highly relevant to disease states affecting light detection by the eye. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The long-term objective of this proposal is to understand quantitatively the transduction mechanism in olfactory sensory neurons (OSNs). At present, there is hardly any biophysical information about each step in the transduction process. This information is crucial for truly understanding the process and ultimately its disease states, as amply exemplified in the advancements in our knowledge about retinal phototransduction in the past two decades. The specific aims are: 1) To examine the interaction between the odorant and the receptor protein, and to find out whether odorant receptors have any constitutive activity in the absence of odorants. 2) To understand the nature of the unitary event underlying the macroscopic olfactory response, and to see whether the response of an OSN to a single odorant molecule is indeed detectable. We shall also dissect the contributions of the cyclic nucleotide-gated current and the Ca-activated CI current to the unitary response. 3) To quantify Ca-dependent adaptation of the olfactory transduction process, and 4) To study Ca-independent adaptation of the process, and the possible roles of GRK3, RGS2 and phosducin in the termination of the olfactory response. The experimental approach involves recording from single OSNs from frog and mouse with a suction pipette. Genetically engineered mouse lines will also be used.

DESCRIPTION (provided by applicant): The long-term objective of the proposed research is to understand in detail the phototransduction mechanism in retinal cones. The specific aims are: 1) to measure the rate constant of spontaneous isomerization of human red cone pigment with 11-cis retinal as chromophore in a transgenic mouse line, in order to assess its importance in human cones. 2) to measure and analyze the response properties and background-adaptation behavior of goldfish red, green and blue cones and to correlate these parameters with the separately measured dark phosphodiesterase activity in each cone type (from which the spontaneous isomerization rate of each pigment type can also be obtained). Goldfish retina has the advantage that it has all three cone types like human retina, and the correlate of biochemical studies by others. 3) to examine the 50-year-old question of whether there is a strict quantitative relation for various pigments between the lambda-max of the action spectrum and the activation energy of photoisomerization, and to correlate this information with the energetics of the associated spontaneous isomerization activity in order to firmly establish photoisomerization and thermal isomerization as fundamentally the same process. 4) to examine the contributions of the pigment kinase (GRK1 and 7), arrestin, and the GAP protein RGS9 to the response properties of cones. We shall employ morpholinos to knockdown each transduction protein in turn in Xenopus and see how this affects the cone response to light. The proposed work will lead to a much greater understanding of the light response of cones, which are more important than rods for human vision. The gained knowledge will also help understand and treat human diseases affecting cone function.

PROJECT SUMMARY The long-term objective of this proposal is to understand in quantitative detail the transduction mechanism in vertebrate olfactory receptor neurons (ORNs). During the current grant period, we have been able to resolve the response triggered by a single odorant-binding event on an ORN (the so- called unitary response). Surprisingly, we found that randomly encountered ORNs (and therefore randomly encountered odorant receptors, or ORs) all gave unitary responses of similar amplitudes, suggesting that an odorant-binding event has a very low probability of signaling downstream. In other words, the unitary response appears to reflect the effect of a single activated Golf/adenylyl cyclase complex. This phenomenon is apparently due to a very short odorant dwell-time on the OR molecule (i.e., rapid dissociation of the odorant from the OR). This short odorant dwell-time likewise dominates the termination of olfactory transduction. Hence, the traditionally believed determinants for terminating G-protein-coupled receptor (GPCR) signaling, namely, receptor phosphorylation and subsequent arrestin binding, are unimportant at least at the level of the unitary response, although they may still be important with intense and prolonged odorant stimulation. So far, the above results have been obtained from amphibian ORNs and in low-Ca2+ solution (in order to boost the unitary-response amplitude). In this application, we propose to continue experiments with amphibian ORNs but at the same time to use also mouse ORNs, which offer the distinct advantage of genetic manipulations. Aim 1 is to obtain the best estimate of the olfactory unitary-response amplitude in frog ORNs in physiological conditions (i.e., normal external Ca2+), to characterize its spatial spread along the olfactory cilium, to dissect its membrane-current components (cyclic-nucleotide-gated current versus Cl current), and to estimate the number of unitary responses required for bringing the ORN to firing threshold. Aim 2 is to launch a similar detailed study of the unitary response in mouse ORNs, which will serve as the groundwork for examining available genetic mouse lines for olfactory transduction. Aim 3 is to study/dissect Ca-dependent and Ca-independent adaptation by ORNs in more detail, largely by making use of genetic mouse lines. Aim 4 is to study constitutive OR activity in the absence of odorants, which so far has received little attention in vertebrate olfactory transduction. We have noticed this phenomenon in the course of previous experimentation. The experimental approach will involve predominantly suction-pipette recording from single ORNs of frog and WT or genetically engineered mice.

DESCRIPTION (provided by applicant): Besides the rod/cone pathway, a melanopsin-associated photoreception pathway is now known to exist in the retina. This pathway involves melanopsin-expressing retinal ganglion cells (mRGCs), a small subset of RGCs that are intrinsically photosensitive but also receive synaptic inputs from the rod/cone pathway. Unlike the non-mRGCs, which project predominantly to image-forming visual centers in the brain (the dorsal lateral geniculate nucleus and the superior colliculus), the mRGCs project primarily to non-image-forming (accessory) visual centers such as the suprachiasmatic nucleus and the intergeniculate leaflet (for circadian photoentrainment) as well as the olivary pretectal nucleus (for pupillary light reflex). Thus, there appears to be a functional segregation between non-mRGCs and mRGCs, with the mRGCs not only contributing to non-image-forming photoreception but also constituting the major conduit for all non-image-associated photic information to the brain. Besides the rod/cone system and the melanopsin system, there appears to be no other photoreception system in the eye that signal to the brain. The long-term objective of this proposal is to understand the light responses and the signaling by mRGCs in great detail. Aim 1 is to investigate the general physiological and biophysical properties of the light responses of mRGCs in detail. Aim 2 is to investigate light adaptation by mRGCs. Aim 3 is to investigate the conversion of the receptor potential into action potentials in the mRGCs, and the roles played by the various voltage-gated ion channels. Aim 4 is to understand the phototransduction mechanism in mRGCs and to identify the various proteins involved in this process. Both electrophysiology and mouse genetics will be used for study. These experiments will yield important information not only relevant to the basic understanding of this newly discovered melanopsin system, but also to disease states affecting light detection in the eye. PUBLIC HEALTH RELEVANCE: The studies proposed in this application will enhance our understanding of the newly discovered melanopsin-associated photoreception system in the retina. Any new information derived from these studies will also be highly relevant to disease states affecting light detection by the eye.

It is now clear that the mammalian retina has at least one other photoreceptor class besides rods and cones, consisting of a subpopulation of retinal ganglion cells (RGCs) that express the pigment melanopsin (OPN4) and are intrinsically photosensitive (ipRGCs). These cells project predominantly to non-image-vision centers in the brain, serving functions such as circadian photoentrainment and pupillary light reflex. At the same time, ipRGCs project moderately to the brain's image-vision centers, presumably serving subtle image- forming visual functions such as possibly providing information about absolute light intensity in the visual scene. IpRGCs comprise at least 5 subtypes, M1-M5, which differ in the level of melanopsin content, photosensitivity, somatic and dendritic-field size, exact locations of their dendritic arborizations in the retina's inner plexiform layer, and the detailed locations of their axonal projections in the respective brain targets. To fully understand the ipRGC system, it is first and foremost important to know in detail their light- response properties and the underlying mechanisms. In recent years, we have made great progress in understanding M1-ipRGCs, including the molecular identities of several key phototransduction components. Specifically, this signaling pathway closely resembles in motif that found in fly-eye photoreceptors. Recently, we have made the exciting discovery of yet another phototransduction pathway in ipRGCs. This grant application constitutes a continuation of these successful investigations. Aim 1 is to establish the details of the second phototransduction pathway discovered by us, and to know the relative importance of the two signaling pathways. We would also like to complete the phototransduction pathway in M1-ipRGCs by establishing the molecular identity of the G protein involved. Aim 2 is to establish the details of light-response termination in ipRGCs, focusing on potential melanopsin phosphorylation, arrestin binding to melanopsin, and G-protein deactivation. Aim 3 is to study the intensity-response relation and the response kinetics of M2- and M4-ipRGCs, to detect/estimate their single-photon-response amplitudes and the melanopsin density in the membrane, as well as to measure their respective light-signaling threshold in order to assess their efficiency in signaling absorbed light. The overall goal is to compare/contrast M1- (already known), M2- and M4-cells. These properties may have differential significance with respect to the specific functions and neuronal circuitry associated with the different subtypes. In summary, ipRGCs are important for non-image vision and subtle aspects of image vision. Therefore, learning in detail how they function is of fundamental importance to vision in both health and disease.

? DESCRIPTION (provided by applicant): The long-term objective of this proposal is to understand in quantitative detail the cAMP- and Ca2+-signaling in sensory transduction by olfactory receptor neurons (ORNs). We shall focus on the question of canonical olfactory-transduction mechanism in ORNs of the vertebrate main olfactory epithelium. The major chemo-transduction mechanism in ORNs of the main olfactory epithelium involves a cAMP-signaling cascade, leading to Na+ and Ca2+ influxes through a cyclic-nucleotide-gated (CNG), non-selective cation channel to depolarize the ORN to firing threshold. The Ca2+ influx leads to signal amplification via a Ca2+-activated Cl channel, as well as olfactory adaptation via multiple Ca2+-activated negative-feedback pathways. Recently, the significance of these amplification and negative-feedback pathways is nonetheless thrown into doubt and confusion. We propose to refine and quantify our understanding of the transduction process and to resolve the confusion surrounding the signal amplification and negative feedbacks. In Aim 1, we shall characterize the ORNs from the M71-monoclonal-nose mouse line, with the objective of using it as the common genetic background for many of the experiments in this proposal. This genotype offers the distinct advantage of having a near-homogeneous population of ORNs in the main olfactory epithelium, making experimental investigations much more efficient, and, in some cases, exclusively possible. In Aim 2, the objective is to carry out a detailed dissection, in both amplitude and time, of the overall ORN olfactory response into its CNG and Cl- current components for determining their correlations in amplitude and kinetics. In Aim 3, our goal is to re-examine the hypothesis that the Ca2+-mediated signal amplification via the Ca2+-activated Cl- current dictates the stimulus threshold for odorant detection. In Aim 4, our objective is to resolve the mystery/confusion about the relevance of the Ca2+-mediated negative feedbacks in olfactory transduction. Elucidating the steps of olfactory transduction will provide great insight into olfactory malfunctions arising from genetic defects in the transduction pathway, as amply demonstrated by the huge success in the case of vision.

PROJECT SUMMARY/ABSTRACT Circadian rhythms are the near-24-hour rhythms of physiology ubiquitous to almost all eukaryotic life. Dysfunction of circadian rhythms underlies a variety of common sleep disorders and is thought to contribute to other conditions ranging from psychiatric disease to cancer. The mammalian retina serves a critical function in synchronizing the master circadian pacemaker (the suprachiasmatic nucleus) to the daily light-dark cycle. Work over many years has also demonstrated that the retina itself is a strong circadian oscillator. Indeed, many critical retinal functions, including visual sensitivity, the pupillary light reflex, the electroretinogram, and the expression of hundreds of retinal genes, are under strong circadian control; and that loss of retinal circadian rhythms results in impaired retinal function. Our preliminary data have demonstrated that: 1) the retinal circadian clock can be entrained to light- dark cycles in culture ex vivo, 2) this entrainment is not dependent on the classical rods and cones or the melanopsin-expressing, intrinsically-photosensitive retinal ganglion cells, 3) the orphan opsin neuropsin (OPN5) is necessary for this photoentrainment, and the orphan opsin encephalopsin (OPN3) affects this process, 4) the retina utilizes a light-dependent, diffusible substance to synchronize its rhythms, and 5) the cornea also contains a circadian clock which, remarkably, can be entrained to light- dark cycles as well via an OPN5-dependent mechanism. We propose experiments to elucidate the signaling mechanisms of OPN5 and OPN3; experiments to characterize the diffusible signal(s) emanating from the retina, and experiments to elucidate the mechanism by which non-retinal tissues in the eye maintain circadian rhythmicity and entrain to light-dark cycles. These data will provide a critical basis for understanding how the circadian clock modulates retinal function as well as mechanistic insights into two novel ocular photoreceptors.