Timothy W. Kraft - US grants

The transformation of an image on the retina into a perception of physical objects begins with the transduction of photon energy into electric signals in rod and cone photoreceptors. Prior to leaving the eye the signals of the -100 million photoreceptors are compared, compressed, averaged, analyzed and reduced into 1 million channels of information flowing in the optic nerve. These ganglion cell nerve fibers carry the information of form, brightness, color and motion encoded as action potentials. Photoreceptors were once thought to act as independent light sensors, like the individual grains of a photographic film, but this appears not to be true. In fact the photoreceptors, the first layer of nerve cells containing visual information, are in contact with one another and therefore may also be responsible for the first level of visual information analysis by the nervous system. There are two hypotheses that will be tested with this grant: Hypothesis I: The subtypes of human red and green cone pigments are expressed randomly one gene per cone cell. A direct examination of human cone spectral sensitives will be compared to the analyzed sequences of the visual pigments proteins. Electrical recording will give the precise descriptions of the spectral sensitivity functions and in parallel the number and variety of visual pigment proteins expressed will be determined. Specific questions are: What ate the spectral sensitivities of human cones? Are multiple copies and varieties of the visual pigment genes functionally expressed of visual pigment genes limited to one per cell? Hypothesis 2: Spectral classes of cones are distinct cell types, that form coupled networks which exclude other spectral classes of cones. Physiologic and anatomic techniques will be used to evaluate of the size and spectral characteristics of a cone's receptive field, this entails a quantitative determination of the strength of synaptic contact between neighboring cones. The presence of three spectral classes of cones in humans raises the issue of how the eye maintains the integrity of its three color "channels" when pooling of information is likely to occur even within the photoreceptor layer itself. The following questions will be approached: (1) Ate mammalian cones eclectically coupled to their neighboring photoreceptors? (2) Are synaptic interactions with neighboring photoreceptor limited to those of the same spectral type? (3) What is the lateral spread of excitation in human and non-human mammalian cones, and (4) are the physical dimensions of the lateral processes between neighboring photoreceptors quantitatively related to the physiologically measured space constant of the cell?

Retinal degeneration is a major cause of blindness in our elderly population as well as the tens of thousands of younger Americans afflicted with inherited retinal degenerations such as retinitis pigmentosa (RP). Photoreceptor degeneration is a final common pathway resulting in loss of vision for many insults to the eye, including many mutations of rhodopsin or other proteins of the phototransduction cascade. In retinal degenerations caused by mutations in rod-specific genes, it is equally important to comprehend why the normal cone photoreceptors also die bringing patients from night blindness to near total blindness. We propose experiments on two newly characterized animal models of autosomal dominant retinitis pigmentosa, the transgenic pig carrying mutant rhodopsin. This grant will answer three important questions: (1) How do the P347L and P347S rhodopsin mutations alter normal phototransduction and rod signaling? (2) How do electrophysiological recordings of single cell photoresponses compare to those same responses derived by indirect methods with the electroretinogram (ERG)? This project will document the ERG's capacity as a tool used to probe photoreceptor function. Massive loss of rod photoreceptors in these animals and in patients with RP somehow kills the cone photoreceptors as well. If cone function could be rescued, a substantial portion of human visual behavior would remain intact. (3) What are the pathologic changes in the physiology of cone photoreceptors associated with retinal degeneration due to rhodopsin mutations in the pig? The past decade has seen tremendous advances in the understanding of the biochemistry and molecular biology of phototransduction, yet little is known about photoreceptors pathophysiology. We will investigate the single cell photocurrents with the suction electrode technique to examine the changes that take place throughout the course of the retinal degeneration and loss of vision. Several biophysical parameters of the rods and cones will be measured at 3 to 5 stages over a period in which all the rods and half the cones are lost. We will also examine the photoreceptor responses to flickering light which is predicted to be a sensitive indicator of cell health. Statistical analyses will determine major and minor effects. We will establish a quantitative physiological database for photoreceptor function in a degenerating retina coordinated with ERG evaluations of the retina at the same stages of disease. This data base will be useful in judging therapeutic intervention.

DESCRIPTION (provided by applicant): The retina contains one of the best-studied G-protein coupled receptors (GPCR), the light-activated phototransduction cascade responsible for visual perception. As such it serves as a model for GPCR systems throughout the body, and yet there remain important unanswered questions about the feedback and modulation of GPCR signaling, especially as it affects termination of the cascade. The reliable shutoff is highly controlled in photoreceptors, and it establishes the temporal capabilities of the visual system. More importantly, photoreceptors can alter shutoff timing as an adaptation to changes in ambient illumination. These molecular events are understood in part, but further work is required to define the multiple mechanisms of adaptation. The first goal of this proposed research is to identify the conditions that elicit a newly discovered form of adaptation in rod photoreceptors and to ascertain the underlying mechanisms controlling it. This form of adaptation reveals a paradoxical hypersensitivity of our dim light photoreceptors following exposure to bright light. We propose to study this rod adaptation at cellular level as well as through clinical and behavioral tests in human subjects. This work will reveal new information about visual system adaptation during mesopic lighting conditions, the twice-daily twilight evolutionarily vital to bot the hunter and the hunted. Additionally, our efforts will provide a more thorough understanding of how the GPCR pathway can be modified to adapt to constant stimuli. Despite being the minority cell in the human visual system, cone photoreceptors have tremendous importance for daytime vision, spatial acuity, and color perception. At this point, there is a far better understanding of rod mechanisms, many of which do not directly translate to cone function. One important aspect that needs to be investigated is the immense adaptive range of cones and the speed with which they accomplish the task. Indeed, the temporal flicker resolution of cone vision measured behaviorally needs to be explained in more detail on the single cell level. A more complete understanding of cone function and adaptation will further not only the field of vision, but the entire field of neurobiology with respect to different GPCR signaling pathways.

UAB Center Core for Vision Research - Ocular Phenotyping Core Project/Summary Abstract Assessment of ocular structures and visual function is essential to both basic and translational research in vision science whether in animal models or human subjects. Widely used technologies include optical coherence tomography (OCT) for display of layered tissues in posterior and anterior segments, electroretinography (ERG, for massed retinal signals separable into multiple identified components), slit lamp for bio-microscopy of anterior and posterior segment, imaging of the fundus via multiple modes of visualization (color, autofluorescence, dye- based angiography, infrared reflectance), and optokinetic nystagmus (to assess visuomotor control, visual acuity and contrast sensitivity). In response to growing UAB vision researcher needs, the ?Ocular Phenotyping Core? was established to encompass a comprehensive suite of instrumentation and to provide the necessary support for accurate ocular phenotyping. Specific instruments include Bioptigen 840 nm SD-OCT and Micron IV digital fundus camera for small animals, Optomotry optokinetic nystagmus in small animals including rats, mice and zebrafish, and Spectralis SDOCT for large animals and human donor eyes. An S10 grant awarded to Dr. Paul Gamlin in 2020 has allowed the purchase of three new tools that will greatly enhance ocular phenotyping of both large and small animal tissues 1) Zeiss Lumera 700 ReScan with Resight 700 operating microscope with intraoperative optical coherence tomography (OCT) imaging; 2) FLEX Module Spectralis OCT2 System for imaging the retina and optic nerve head with structural and angiographic OCT, in animals at various body positions; 3) Anterior Segment CASIA SS-1000 swept source OCT (Tomey Corp.) for imaging the cornea, iris, and lens in large animals, as well as the whole eye in small animals. This core will support 15 UAB Vision Scientists, including 13 with planned moderate to extensive all of whom are currently NEI R01-funded. The Director and Associate Director of this proposed core have extensive publication experience in electroretinography and OCT validation/ interpretation, respectively. New directions for the core will include the completion of a new LED-based ERG system that will simplify this testing. Further, an existing AOSLO system originally designed for human retinal imaging will be re-engineered in order to image rodent and tree shrew eyes in vivo. At the same time, capabilities for fluorescence imaging and cell-targeted photo-stimulation will be added. Additionally, Ocular Phenotyping Opportunities will be advertised to identify new ocular mouse models through full ocular phenotyping screens of mouse models generated by non-ocular scientists whose animals were originally generated to answer questions pertinent to their organ systems of interest.

Cone photoreceptors in the retina mediate the vast majority of day-to-day visual percepts in humans, and their responses represent the initial visual signals processed by downstream neurons. As a result, characterizing the response properties of human cone photoreceptors is key to addressing gaps in our knowledge of retinal physiology, and ultimately, the neural basis of human vison. Most studies of mammalian cone electrophysiology have been conducted in vitro. Although this allows extensive control, evidence suggests differences exist in cone behavior under in vivo and in vitro preparations. To approach this issue in vivo, our lab developed a technique in which cone response properties can be elucidated in vivo using single-cone targeted retinal stimulation with an adaptive optics scanning laser ophthalmoscope (AOSLO) in conjunction with physiological recordings from downstream neurons. In addition to using normal single photon stimuli, we will utilize 2-photon stimuli to examine the nature of direct 2-photon stimulation of cone photopigment using the AOSLO system. Though there exists evidence suggesting direct 2-photon stimulation of cones occurs (Palczewska et al. 2014), there remains the possibility that such percepts arise from secondary fluorescence of endogenous retinal fluorophores. By interleaving single and 2-photon stimulation of cones, we will also be able to quantify the differences in light capture between the two stimuli. Data gathered over the course of this project will also be used to examine neural encoding in the optic tract and the lateral geniculate nucleus (LGN) as it relates to psychophysical luminance threshold. Because LGN neurons receive many more spikes than they produce, the LGN must selectively relays certain spikes (Sincich et al. 2007, Rathbun et al. 2010). How the LGN spike patterning at a given stimulus detection probability leads to perceptual threshold is not understood, particularly in response to single cone stimulation. The goal of this proposal is to characterize the response properties of macaque cone photoreceptors in vivo and determine if spike coding in retinal ganglion cells or the LGN set the bounds for human psychophysical luminance threshold performance. In Aim 1, we will use cone-targeted stimuli to measure cone intensity response functions, time course of recovery to a flash, and adaptation to repeated stimuli. By incorporating 2-photon stimuli, we will quantify the extent of indirect excitation of cones when nearby endogenous fluorophores emit light following 2-photon excitation. In Aim 2, analysis of the spike trains recorded in Aim 1 will be used to determine how the neurometric response function relates to psychophysical response functions. The findings of this study will address fundamental gaps in our understanding of neural activity in the early visual system and the mechanisms underlying 2-photon perception.