David R. Copenhagen - US grants

This research is an investigation of synaptic interactions in the retina of the eye. More specifically, this research will concentrate on synaptic processing in the outer plexiform layer with an emphasis on identifying the synaptic transmitter substance released by photoreceptors and determining the actions of this transmitter. This study will use a newly developed technique called noise analysis. This analysis will allow characterization of how the postsynaptic channels in bipolar cells are affected by the photoreceptor transmitter. Putative transmitters will be superfused onto the retina and the noise analysis technique will again be used to characterize properties in bipolar cells. Comparing the properties of the channels under the normal and perfused states will allow us to identify the natural transmitter. A second part of this study will involve the fabrication of substrate specific electrodes which will be employed to measure directly the release of neurotransmitter from retinal neurons. This technique involves the use of pH or NH+ detectors coated with a gelatin which is embedded with an enzyme that degrades or hydrolyzes the neurotransmitter producing NH+ or H+ ions.

The long term goal of this proposed work is to elucidate the biochemical, anatomical and electrophysiological basis of vision. Emphasis will be on the functioning of the neural retina with a primary goal of understanding how the photoresponses generated by the rods and cones are synaptically transmitted by way of two or more intermediary classes of neuron to the ganglion cells. The secondary long-term goal is to develop an understanding of the pathophysiology of retinal disease processes and inborn errors of metabolism. The approach is an experimental one that focuses at the cellular level. Microelectrode techniques will probe the electrical behavior of individual neurons and the biochemical processes of synaptic transmission. The specific aims of this project period are to identify the transmitter substance released by photoreceptors and to determine by what biochemical and pharmacological means this release is controlled and regulated. For this purpose we intend to develop methods to detect directly the transmitter substance as it is secreted from the rods and cones. A second aim of this project period is to determine the action that the photorecptor neurotransmitter has on the second order neurons in the retina. Among the actions to be identified are the types of postsynaptic receptor proteins activated by the neurotransmitter, and the ionic specificity, voltage dependencies, and kinetics of the conductance channels activated by the neurotransmitter. A third aim is to elucidate the synaptic mechanisms that excite and inhibit the ganglion cells. The ganglion cells, being the output neurons of the retina, integrate the information coming from the more distal parts of the retina. The pharamcology and biophysical parameters of the synaptic inputs to the ganglion cells will be investigated.

The overall goal of the research is to determine how light-evoked signals in the retina are formed and how they are modified by neural activity. The neural image of the visual scene that is processed by the retina and conducted to brain is the result of a complex interplay between excitation and inhibition. One source of inhibition in the retina is the input from horizontal cells onto photoreceptors and bipolar cells; another source is the input from glycinergic and GABAergic amacrine cells onto ganglion cells. In this proposed study the focus will be on characterizing the generation and modulation of inhibitory activity in these two pathways. The general approach is to characterize first the pharmacological and biophysical bases of cell-cell interactions and then incorporate these mechanisms into a more general model. For horizontal cells the study will be directed to investigating the mechanisms by which extracellular neurotransmitters, such as glutamate and GABA, and intracellular second messengers, particularly H+ ions, regulate function. For ganglion cells this project will characterize the action and mechanisms of glycinemediated inhibition in the tiger salamander and mouse retina. The study will utilize patch pipettes to perform voltage-clamp measurements from enzymatically-isolated retinal neurons. These same recording techniques will be used to measure light-evoked responses from neurons in the retinal slice preparation. Intracellular H+ and calcium ion activities will be monitored optically with selective dyes.

9315127 Janecke This award supports a two-year cooperative research project between Professor Joachim Janecke, Department of Physics, University of Michigan, and Professor Mamoru Fujiwara, Research Center for Nuclear Physics, (RCNP) Osaka University, Osaka, Japan. In addition to Professor Janecke, a postdoctoral fellow and a graduate student from the University of Michigan will visit Japan to participate in the joint research project. The Michigan researchers will perform experiments related to the study of charge-exchange reactions and the properties of giant resonances at the new Ring Cyclotron Facility and the new magnetic spectrometer Grand Raiden at Osaka University. The particle energy available at the RCNP facility favors spinflip transitions. The proposed experiments to investigate microscopic structures of Gamow-Teller resonances and characteristics of the charge-reaction mechanisms in terms of isovector non-spinflip, spinflip, and tensor interactions represent an extension and continuation of work performed by the U.S. principal investigator at the Indiana University Cyclotron Facility over the past several years. These experiments will yield new data that may shed light on the character of isobaric analog states. ***

The detection and signaling of visual information in retina requires the establishment and maintenance of millions of individual synapses. Not only must these synaptic circuits reliably and rapidly transmit the light-evoked signals generated in rods and cones, they must be modifiable as lighting conditions change during the day. Excitatory signals in the retina, and the brain, are transmitted via synapses that use glutamate as their neurotransmitter. Because glutamate does not cross the brain/blood barrier, glutamate has to be synthesized in the nervous system. A glutamate/glutamate recycling system replenishes synaptic glutamate. In retina, little is known about how glutamine is transported out of the Mtiller glial cells and back into the glutamatergic neurons for re-synthesis of glutamate. A principal aim of proposed research centers on identifying and characterizing the glutamine transport mechanisms in retina. Recently it has been discovered that glial cells are not passive elements compared to neurons but they release neurotransmitters that regulate synaptic transmission between neurons. A second aim of this research is to identify and characterize the mechanism(s) by which glutamate is released from Muller glia cells in retina. Elucidating these mechanisms in the retina, a more experimentally tractable region of the nervous system, will serve as a model for glutamate regulation in the CNS. Glutamate-induced excitotoxicity underlies many of the changes produced by pathological conditions such as ischemia and glaucoma. Neurotrophins regulate development and maintain synaptic activity. Examination of mice lacking the neurotrophin receptor TrkB show decreased synaptic transmission from rod photoreceptors. To eliminate deleterious effects that TrkB deletions have on other somatic functions Louis Reichardt will use retina-specific promoters to delete TrkB receptors in the eye only. A third major goal is to characterize the effects of these deletions on light-evoked responses in retina.

PROJECT SUMMARY/ABSTRACT The goals of this research are focused on understanding the signaling mechanisms that regulate the development of neural circuits and visual function in the maturing neonatal retina. Our own previous work, as well as that of others, has demonstrated that visual deprivation can suppress the normal maturation of dendritic arbors and the temporal properties of light responses of ganglion cells in the inner retina. We have found that the neurotrophin BDNF (Brain-Derived Neurotrophic Factor) is down regulated by visual deprivation and it plays a critical role, through activation of the TrkB receptor, in governing the refinement of more diffuse dendritic arbors of ON-OFF type ganglion cells into more narrowly stratified arbors that are restricted to ON and OFF synaptic regions of the inner plexiform layer of the retina. Other studies in brain have demonstrated that BDNF/TrkB signaling also regulates the formation of inhibitory synapses using GABA as a neurotransmitter. Because GABAergic neurotransmission plays such a critical role in visual processing in the retina we are proposing to test the hypothesis that BDNF/TrkB regulates the maturation of GABAergic circuits in the retina. Excitatory neurotransmission is also required for visual signaling in the retina. The excitatory inputs to retinal ganglion cells are mediated by glutamate release from bipolar cells that concomitantly activates two types of glutamate receptor, the NMDA- and AMPA-type. NMDA-type receptors are not only important for rapid signaling of neural signal but also play fundamental roles in the development of neuronal structures, the formation of functioning synapses and the modification of synaptic strength. NMDA receptor expression in the retina is itself regulated by light suggesting the activation of these receptors might, in turn, govern the maturation of synaptic connectivity and synapse function in the retina. We will be directly testing the hypothesis that NMDA receptors regulate the refinement of ganglion cell dendrites and the insertion of AMPA receptors into synaptic sites. We will employ genetic and pharmacological method to block both BDNF/TrkB signaling and NMDA receptor expression and function. In a final aim we will test the hypothesis that melanopsin expressing ganglion cells mediate a visual behavior, called negative phototaxis, in very young neonatal mice. We have preliminary evidence that the melanopsin containing ganglion cells, which are independent of rods and cones and are light responsive well before rod and cone visual signaling begins, are the photodetectors for this early visual behavior. Although melanopsin ganglion cells are known to control pupil responses and photoentrainment of circadian rhythms, this proposed early neonatal function for melanopsin has not been described previously. These experiments are designed to provide insight to how the visual system develops, what processes might lead to dysfunctional vision and what compensatory visual mechanisms might be functioning in blindness when rod and cone function is compromised.

SUBPROJECT ABSTRACT NOT PROVIDED

DESCRIPTION (provided by applicant): We propose to continue a successful Training Program for predoctoral students and postdoctoral fellows in the visual sciences. The core of the training program is coupled strongly with and through the Neuroscience Graduate Program at UCSF. The emphasis will be on training students and fellows who will apply modern biological methods to research problems in the visual sciences. All trainees will receive comprehensive research training in the laboratory of their mentor. Additionally, trainees will receive a strong foundation in visual sciences and the disciplines of modern biology through formal course work, seminars, journal clubs, and contacts with the clinical and basic science faculty at the UCSF School of Medicine. All of the faculty members of the Training Program are established scientists who are members of the Neuroscience Program. Most of the faculty members also have affiliations with other graduate programs at UCSF. Eight of the training faculty are also members of the Department of Ophthalmology, and seven of these have neighboring laboratories in the Koret Vision Building. Twelve of the faculty members receive their primary research funding from the National Eye Institute. The research interests of the entire faculty encompass or complement one or more areas of the visual sciences. Predoctoral trainees will be selected after choosing a research mentor and following completion of the Neuroscience Core courses and 3-laboratory research rotations. The progress of predoctoral trainees will be monitored by a thesis committee and by the Vision Training Program. Postdoctoral fellows will join and train primarily in the laboratory of a selected faculty member. Collaboration with the laboratories of other training faculty, or other faculty within the School of Medicine, will be strongly encouraged. Such collaborations are a strong tradition at UCSF. UCSF is particularly well suited for this Training Program because of the strength of its research and graduate training in molecular biology, cell biology, molecular genetics, and neurosciences, and because of the quality of broad-based research in the visual sciences. The exemplary record of successful training of predoctoral students and postdoctoral fellows by the mentors on the Vision Training Grant shows the capabilities and dedication of our program. Of the 78 postdoctoral fellows who have completed training in the labs of the Vision Training Faculty in the last 10 years, 57% are presently in independent academic positions at universities, medical schools and dedicated biomedical research institutes such as the Riken in Tokyo. Another 14% remain in research in an academic setting and another 14% have taken an industrial position in companies associated with biomedical products. Continued NIH support for this training grant will be essential for enabling us to continue training scientists and academicians for the future. PUBLIC HEALTH RELEVANCE: The goal and mission of biomedical research, and NIH, is first to search for and discover fundamental knowledge about the nature and behavior of living systems and then to apply that knowledge to enhance health, lengthen life, and reduce the burdens of illness and disability. The goal of predoctoral and postdoctoral training at UCSF is to guide and educate future researchers who will make fundamental creative discoveries, develop innovative research strategies, and apply their skills and knowledge as a basis for ultimately protecting and improving health. Additionally, this training is organized to promote the highest level of scientific integrity, public accountability, and social responsibility in the conduct of science. Because we are training students and fellows in the visual sciences, we will be focused ultimately on the causes, diagnosis, prevention, and cure of human blindness. Given the research interests and expertise of the faculty members in this program, predoctoral and postdoctoral training will also emphasize the growth and development of the visual system. This proposed training program will play an important role in educating students who will become our future biomedical researchers. Having a well-trained cadre of researchers is the principal means that we, as a nation, have for understanding and treating illnesses and disabilities of our own and the world's population.

DESCRIPTION (provided by applicant): Light is an all-pervasive biological stimulus. Most light on our planet comes from the sun and this is closely reflected in the mechanisms of light response that have evolved. Whether it is the phototropism of a unicellular green alga, the circadian entrainment of plants or high-acuity vision in humans, light elicits a huge variety of biological responses that are of fundamental importance. In this application, we provide evidence that in the mouse (and thus, probably in the human) light is an important stimulus for eye development. Unexpectedly, we found that dark reared neonatal mice exhibit abnormal vascular development in which the regression of hyaloid vessels is retarded and the growth of retinal vessels is promiscuous. The same abnormal growth is observed in light reared mice lacking the photopigment melanopsin. We have strong preliminary evidence that the maldevelopment of the ocular vessels is associated with (1) changes in the retinal level of the neurotrophin BDNF, (2) increased neuron number, and (3) altered VEGF expression level via oxygen demand. Also, surprisingly, the maximal effect of dark rearing is found in pups raised in darkness from E16. Our Central Hypothesis is: Ocular development is regulated by a fetal light-melanopsin-BDNF pathway that regulates neuron number, VEGF expression, and ultimately, postnatal vascular patterning. We propose three experimental aims: (1) To establish whether light activation of the melanopsin pathway in the fetus is necessary and sufficient to regulate vascular development in the eye, (2) to determine how BDNF signaling integrates with light-dependent vascular development, and (3) to define the role of oxygen demand in the light response pathway.

? DESCRIPTION (provided by applicant): A separate non-rod, non-cone visual system is now known to exist and function in the retinas of most mammalian classes from mice to humans. This secondary system is comprised of a subset of retinal ganglion cells in the eye that express the photopigment melanopsin (mRGCs). In adults, activation of these mRGCs regulates photo entrainment of circadian rhythm and pupil constriction. Additionally, these mRGCs are thought to be involved in photophobia during migraine headaches and seasonal affective disorder (SAD). Surprisingly, melanopsin is expressed very early during embryonic development (gestational week 8 in humans and embryonic day (E) 11 in mice). These early born mRGCs are functional well before the maturation of vision that is mediated by the more conventional rods and cones. As evidence, light activation of mRGCs in young neonatal mice evokes photoaversion and vocalizations associated with stress. Moreover, a study of vascular development in mouse eye demonstrated that light exposure of fetuses in utero modulates the timing of angiogenic maturation in the eyes of the subsequently born pups. The effects of light require the expression of melanopsin in the fetal eye. Thus, this strongly suggests that melanopsin expressing cells in the fetal eye can be activated by light exposure and this activation, by signaling to other cells within the eye, modulates and triggers developmental vascular and neuronal programs. No studies to date have directly demonstrated light sensitive neurons in the embryonic retina. We have recent preliminary evidence directly proving that light can elicit responses in melanopsin-expressing neurons in embryonic retinas of mice as young as E16.5, 4 days before birth. One aim of this proposed project seeks to 1) characterize the kinetics and sensitivity of the light responses evoked in the embryonic mRGCs, and 2) to determine the ionic and pharmacological mechanisms of phototransduction in these same mRGCs. A second aim addresses intercellular signaling from mRGCs to other cells in the embryonic retina. Given that light activation of these embryonic mRGCs is coupled to pathways regulating vascular and neuronal development in the eye, it is imperative to understand by what means these signals are communicated to other cells in the embryonic retina. This is an important first step in elucidating downstream signaling that can regulate subsequent maturation. These experiments significantly extend the formerly classical concepts of when vision and light sensitivity become manifest in the maturing infant. Because melanopsin expression occurs very early in the human eye, an inference from our mouse studies would argue that premature infants between GW24 and 30 should exhibit melanopsin-mediated photoresponses in their retinas. This provides a basis to attend to photo aversive behavior and light-regulated circadian rhythms in very young, prematurely born infants.