Zhuo-hua Pan - US grants

The regulation of transmitter release at retinal bipolar cell terminals is important for visual signal processing from the outer retina to the inner retina in vertebrates. Although the central role of Ca2+ in transmitter release at presynaptic terminals is well established, the dynamic regulation of intracellular Ca2+ concentrations ([Ca2+]i) at presynaptic terminals and its effect on transmitter release is still largely unclear. The objective of this proposal is to study the roles of Ca2+ channels and GABA receptors in the regulation of Ca2+ dynamics at the axon terminal of mammalian bipolar cells. Experiments will be performed in solitary rat and mouse bipolar cells. Ca2+-sensitive fluorescence dyes will be used. Fluorescence signals will be monitored by a high resolution confocal microscope based imaging system. Cell's membrane potential will be controlled by patch-clamp methods and, alternatively, cell depolarization will be evoked by high K+. The specific aims are: I) to determine the spatial distribution of different types of Ca2+ channels in bipolar cells; 2) to characterize the Ca2+ transients at bipolar cell terminals during the activation of different Ca2+ channels and during cell depolarization that mimics bipolar light response waveforms; 3) to characterize the distinct effects of GABA A and GABA C receptors on the regulation of the Ca2+ transients at bipolar cell terminals; 4) to determine the spatial distribution of GABA receptors and the possible co-localization of specific types of GABA receptors with specific type(s) of Ca2+ channels. The knowledge we gain from these studies will not only lead to a better understanding of basic visual signal processing in mammalian retina but will also contribute to our knowledge of the roles of Ca2+ and inhibitory neurotransmitters in synaptic transmission in the CNS.

DESCRIPTION (provided by applicant): One of the important features of visual information processing in the retina is the conversion of relatively sustained light responses in the outer retina to more diversified responses, including the formation of the transient response, in the inner retina. Increasing evidence suggests that bipolar cells play an active role in retinal processing and that the diversified responses in the inner retina originate, in part, by means of the different forms of transmitter release from bipolar cells. By what mechanism(s) the different forms of transmitter release are generated and to what extent bipolar cell processing contributes to the overall retinal processing remain to be elucidated. The long-term objective of this proposal is to understand the roles of voltage-dependent membrane channels in bipolar cell signal processing. Mammalian retinal bipolar cells express a variety of voltage-dependent channels, including low-voltage-activated (LVA) and high-voltage-activated (HVA) Ca2+ channels at the axon terminals and voltage-gated Na+ channels in a subset of cone bipolar cells. The physiological roles of multiple voltage-activated Ca2+ channels and voltage-gated Na+ channels in bipolar cell signal processing will be investigated. The first part of this proposal is to study the roles of LVA and L-type HVA Ca2+ channels in bipolar cell transmitter release. Specific aim 1 will determine the Ca2+ channel type(s) located at the axon terminals and involved in transmitter release of different subtypes of bipolar cells. Specific aim 2 will determine the temporal properties of transmitter release from bipolar cells during the activation of LVA and L-type Ca2+ channels. The second part of this application is to study the roles of voltage-dependent membrane channels in shaping bipolar cell response waveforms. Specific aim 3 will determine the roles of different voltage-dependent channels in the spontaneous and evoked response waveforms in isolated bipolar cells. Specific aim 4 will determine the subtypes of bipolar cells expressing Na+ channels and the contribution of Na+ currents in bipolar cell light response waveforms. The studies will be carried out with isolated bipolar cells and with bipolar cells in retinal slice preparations of mammalian retinas. Patch-clamp recording and optical Ca2+ imaging methods will be employed. The knowledge we gain from these studies will lead to a better understanding of basic visual information processing in the mammalian retina. The understanding of the role of voltage-activated Ca2+ channels in synaptic transmission may also provide insight for identifying the mechanism of diseases caused by synaptic dysfunction.

[unreadable] DESCRIPTION (provided by applicant): Vision loss or blindness in many human retinal degenerative diseases, such as retinitis pigmentosa and age-related macular degeneration, is a result of the degeneration of photoreceptor cells. At present, there is no real treatment or cure for the blind. Most of the current approaches that are attempting to develop treatments for the blinding disorders are based on two strategies: one involves transplantation of normal or stem cells into the diseased retinas and the other provides electrical-stimulation via retinal implants. We propose a new strategy that aims to restore a certain level of vision for the blind suffering from photoreceptor degeneration. Our strategy is to enable the remaining second or third order neurons in photoreceptor degenerative retinas to respond to light by expressing light-gated channels in their membranes. This strategy is likely to be feasible both mechanistically and technically and can avoid many obstacles encountered by the current approaches. Particularly, directly light-gated micro-type rhodopsins, channelrhodipsins, have been recently cloned and functionally expressed in mammalian cell lines. Studies in blind patients and in animal models suggest that the second and third order retinal neurons remain, in part, preserved in the diseased retinas. Furthermore, it is possible that exogenous genes can be introduced into retinal neurons by means of gene therapy. The objective of this proposal is to conduct feasibility studies for this strategy in rodent models. The specific hypotheses that we will test in this proposal are: 1) directly light-gated channelrhodopsins can be functionally expressed in mammalian retinal neurons, 2) the remaining retinal second and third order neurons in the photoreceptor-degenerative retinas retain their physiological capacity to relay and process visual signals. These studies will establish a foundation for further work leading to the development of a potentially effective treatment for the blinding disorders as well as techniques with valuable applicability in biomedical research. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The severe loss of photoreceptor cells in retinal degenerative diseases often leads to total blindness. At present, there is no available treatment or cure to the blind. Prospective strategies currently being considered for restoration of vision following rod and cone degeneration include transplantation of normal photoreceptor or progenitor cells and direct electrical stimulation of the surviving retinal neurons via retinal implants. We are exploring another strategy to restore light sensitivity to the degenerate retinas: the expression of light-sensitive membrane channels in surviving second- or third-order retinal neurons. This approach is plausible. First, directly light-gated microbial-type rhodopsins, channelrhodopsins, have been recently cloned. Our preliminary results have shown the ability of functional expression of channerhodopsin-2 (ChR2) in retinal neurons in normal and retinal dystrophic animals in vivo and the capability to depolarize the membrane potential of ChR2-expressing retinal neurons by light. Second, studies in blind patients and in animal models suggest that the second- and third-order retinal neurons remain, in part, preserved in the diseased retinas. Furthermore, it is possible that foreign genes can be introduced into retinal neurons by viral-based gene therapy. A potential advantage of this approach is that it does not involve the introduction of tissues or devices into the retina and, thus, may avoid the immune reactions and biocompatibility complications. The objective of this proposal is to address questions that are fundamental to the feasibility and success of this strategy by using rodent models. The specific aims of this proposal are: 1) To examine the longevity and biocompatibility of the expression of ChR2 in retinal neurons in vivo and explore the targeting expression of ChR2 in certain functional sub-populations of inner retinal neurons; 2) To characterize the light-evoked current and voltage responses of ChR2 expressing inner retinal neurons; 3) To examine the physiological properties of the surviving inner retinal neurons after photoreceptor degeneration; 4) To characterize the light response properties in the degenerate retinas following the expression of ChR2. These proposed studies could establish the necessary foundation for further work leading to a potential treatment for blinding disorders as well as power techniques with applicability in retinal research. [unreadable] [unreadable] [unreadable]