John O'Brien - US grants

The long term objectives of this project are to identify the proteins that form retinal gap junctions and to understand the molecular mechanisms of their regulation. Gap junctions, ubiquitous mediators of intercellular communication, play a prominent role in the visual system. Most types of retinal neurons are connected by gap junctions and their modulation during visual adaptation has profound effects on sensitivity and receptive field properties of many neurons and influences the path of signal flow in the mammalian rod circuit. At least two pathways are known to modulate certain retinal gap junctions: a dopamine/PKA and a nitric oxide/PKG pathway. In order to understand fully the regulation of retinal gap junctions, molecular characterization of the gap junction properties and their modulation is needed. We have cloned two perch retinal gap junction proteins, connexins (Cx) 35 and 34.7, which have defined the new gamma branch of the connexin gene family. These are the first connexins to be found primarily in retinal and brain neurons. We now plan to study how the gamma connexins contribute to the gap junctional properties observed in retinal neurons. The general strategy we will use is to identify characteristic molecular and biophysical properties of the connexins in isolated systems and relate them to properties of the gap junctions in the retina. The specific objectives of this proposal are (1) to identify the cells expressing each connexin; (2) to identify and characterize mammalian homologous of the gamma connexins; (3) to examine the differences in permeability properties of the closely related gamma connexins; and (4) to characterize the regulation of the connexins by protein kinases. A variety of biochemical, biophysical, and molecular techniques will be employed to achieve these goals. These studies will provide a detailed analysis of the regulation of this critical group of connexins that play a vital role in the retinal circuitry. The results will lay the groundwork for understanding defects in gap junctional coupling that may lead to neurological disorders.

ABSTRACT NOT PROVIDED

[unreadable] DESCRIPTION (provided by applicant): [unreadable] Aquatic animals are extremely important model systems for research in the life sciences. Decades of research in physiology and biochemistry have depended upon aquatic animals, particularly fish, and many advances in the understanding of fundamental physiological processes and disease processes have come from this research. These well-established model systems continue to be critical components of the research efforts of investigators at the University of Texas Health Science Center-Houston (UTHSC-H).Recent advances in genetic technology have now brought the zebrafish model system to the forefront of research. This powerful model system allows for convenient and economical genetic manipulation in a vertebrate. Several research programs at the UTHSC-H are making use of this model system and faculty recruitment efforts are expected to increase this number. In order to provide support for both the long-established and up-coming model systems, the UTHSC-H will include aquatic animal holdings in its new animal care facility. This project is designed to outfit two rooms in the Animal Care Center, which is currently under construction. The goals of the project are: 1. To establish a central facility for zebrafish maintenance and the development of transgenic fish. 2. To centralize housing and care of freshwater fish species. The first goal will significantly expand current holdings of zebrafish and establish a transgenic facility that can be used by investigators throughout the Health Science Center. The second will shift fish holdings from individual laboratories to the Animal Care Center, and expand capacity. Both will result in improved care and veterinary oversight of aquatic species. These infrastructure improvements will help to accelerate the pace of research and discovery in the health-related fields. [unreadable] [unreadable] [unreadable]

MOLECULAR RESOURCES AND SERVICES MODULE ABSTRACT The Molecular Resources and Services Module provides centralized resources and a variety of specialized services to members of the Vision Research Group to support projects employing cellular and molecular techniques. Core equipment and software supported by the Module is freely available to Vision Research Group investigators, providing most basic needs for molecular work, eliminating the need for redundant equipment and software purchases, and allowing new investigators and investigators initiating new projects to begin work quickly. The Module maintains a well-equipped cell culture laboratory with an experienced technician. Trained investigators may use the laboratory or investigators may use the services provided by the technician to perform experiments with cell lines, primary cells, and cultured tissues. Module faculty provide two high-level services to Vision Research Group investigators. Through a virus development and packaging service we will design and construct lentivirus or adeno-associated virus vectors to deliver gene constructs of interest to animal models. We will also provide a service to validate infectivity and cargo of viral vectors obtained from outside sources. Through a molecular reagent validation service, we will design and perform experiments to validate the specificity of reagents such as antibodies and CRISPR constructs.

Neurons of the central nervous system are organized into networks through a variety of synaptic interactions. Electrical synapses, formed by gap junctions between neurons, are a core component of this organization. Electrical synapses can synchronize activity of networks of the same neuron type and provide rapid, bi-directional electrical communication between neurons of different type. These properties are critical for many high-order network functions of the central nervous system. Electrical synapses are not static, but are tightly regulated. Changes in phosphorylation state of connexin 36 (Cx36) in some retinal neurons modify coupling quantitatively over more than an order of magnitude dynamic range. Such changes in coupling are a stereotyped element of retinal light adaptation, and many electrically coupled retinal networks are held in a very poorly coupled state during the daytime to optimize their function. The vast majority of electrical synapses throughout the central nervous system are composed of Cx36. ALL of these synapses are capable of the same scale of plasticity, but very little is known about the plasticity of most networks or their functional state. This project explores the hypothesis that optimal function of electrically coupled networks is often best served by weak coupling. To test this hypothesis we will develop conditional mutant mice in which the electrical synapses are locked in an open state by point mutations that mimic phosphorylation of Cx36. This should cause unusually high coupling in most networks affected. These animals will be used to determine the impact of strong coupling of neural networks in the retina in the daytime on visual functions and behavior. Further studies will examine specifically the necessity to use electrical synaptic plasticity to reconfigure rod visual pathways in the daytime to optimize ganglion cell responses in photopic light. This animal model will be useful to study plasticity of electrical coupling in most other circuits in the central nervous system. It will have further utility in studies of the role of gap junctional coupling in bystander cell death in brain injuries and diseases.