Stephen C. Massey - US grants

The cholinergic neurons of the rabbit retina, also known as starburst amacrine cells on account of their unique morphology, are a class of excitatory amacrine cells which provide a direct input to certain types of ganglion cells, including the directionally selective group. At the present time, the function of the cholinergic amacrine cells and the role of ACh in regulating ganglion cell firing rate are unknown. The PI proposes to conduct a pharmacological investigation of the cholinergic system in the rabbit retina using a broadly integrated approach which includes: 1) a study of the mechanisms controlling ACh release and 2) recording from individual neurons to evaluate the effects of cholinergic input. Using a well established release technique, the PI will identify the excitatory input to the cholinergic amacrine cells. Since this system is known to receive direct input from bipolar cells, these experiments will also provide information on the identity of the bipolar cell transmitter. Single flash experiments will be used to separate the ON and OFF components in the light-evoked release of ACh. This will permit a comparison of the excitatory and inhibitory inputs to the displaced (ON) and conventional (OFF) cholinergic amacrine cells. By extracellular recording, combined with pharmacology, the PI will compare the light-driven cholinergic input to brisk and complex ganglion cells. Specific experiments will be conducted to investigate interactions between the cholinergic and GABAergic systems. By intracellular recording, the PI will test the hypothesis that only ganglion cells receive cholinergic input. The mechanism of cholinergic excitation may be deduced by measuring changes in input resistance. For directionally selective ganglion cells, ACh and GABA inputs will be isolated and compared. The goal of these experiments is to understand the function of the cholinergic neurons in the rabbit retina. This will be one step toward an understanding of the neuronal circuits which underlie the receptive field properties of retinal ganglion cells.

neural initiation; neuropharmacology; parasympathetic nervous system; neural information processing; amacrine cells; retinal ganglion; gamma aminobutyrate; acetylcholine; neural inhibition; evoked potentials; electroretinography; electrophysiology;

The inner retina is a major site of neuronal processing in the visual system. Here, among a host of amacrine cells, ganglion cell receptive fields are formed, rod and cone pathways merge into one output, and other neurons respond selectively to directional stimuli. Bipolar cells are the principle elements which transmit visual activity from photoreceptors to the inner retina, but neither the identify of the bipolar cell transmitter, nor the output sign have been established. The PI proposes to investigate neuronal circuits in the inner retina using an integrated approach which includes: i) a study of ACh and glycine release to investigate the inputs to cholinergic and glycinergic amacrine cells. ii) extracellular and intracellular recording from the rabbit retina to investigate the function of post-synaptic glutamate recetors. iii) extracellular recording in the primate (monkey or baboon) retina to investigate colour vision and iv) intracellular staining and recording under microscope control. The last technique provides a method to record and stain identified neurons in the living retina, such as the cholinergic amacrine cells. This will simplify the analysis of the mechanism underlying directional selectivity. Glycine, an inhibitory transmitter is found in some bipolar cell which are thought to be excitatory. This anomaly may be explained by the study of dye coupling between glycinergic AII amacrine cells and bipolar cells. Since the gap junctions between these cells are a major site of rod input to cone pathways, the control of gap junction conductance by the dopaminergic cell A18 may act as a switch between rod and cone vision. By dual recordings, the PI will test to see if bipolar cells are inhibitory or excitatory and a pharmacological analysis will be performed to identify the bipolar cell transmitter. The goal of this proposal is to identify the neurons and transmitters of major circuits in the inner retina.

Thirty-five vision scientists at the University of Texas - Houston have formed the Vision Research Consortium which serves as a focal point for support of research in the eye and in the visual system. Substantial administrative support at many levels has been provided for this effort with the overall goal of enhancing research capabilities and collaborative opportunities of basic and clinical vision researchers. The activities described in the grant proposal are an integral part of this institutional-wide initiative. Support is requested from the Core grant for four modules. 1) The Biostatistics Module will provide salary funds for a faculty level biostatistician consultant to enhance the quality of clinical research studies, enhance future collaborations, and develop new research capabilities for prospective research projects. 2) The Confocal and Electron Microscope Module will provide technical support for state-of-the art cellular imaging, using electron microscopy facilities that are already in place, and a new confocal microscope which is being purchased for the Vision Research Consortium by the administration. 3) The Tissue Procurement and Culture Module will upgrade the current culture and tissue storage facilities as well as provide high level technical expertise in culture procedures, and coordinate the procurement of human and primate tissues for culture and other studies. 4) The Laboratory Software Support Module will support a computer scientist to provide training, trouble shooting and consultation for existing hardware platforms such as those used for image processing on the new confocal microscope, and for the development of unique research systems. Support from this grant, coupled with substantial backing from the institution and additional funding from private sources, will provide an environment in which the Vision Research Consortium can reach its full potential as one of the top vision research groups in the country.

tissue /cell culture; eye disorder; vision; eye; biomedical facility;

Vision Research has been identified as an area for investment and development at the University of Texas in Houston. Thus, significant resources, personnel and laboratory space have been committed to the Vision Research Consortium, a faculty group which crosses departmental lines. In the last grant period, the group has grown from 8-12 qualifying NEI faculty members, 5 of whom are now NEI grant holders. These new recruits were attracted, in part, by the outstanding facilities supported by the Vision Core Grant, including a confocal microscope purchased with institutional support. In total, the Vision Research Consortium now consists of 26 vision scientists from the Medical School, the School of Public Health and the M.D. Anderson Cancer Center. Together, they published 210 papers in the last grant period. The members of the Vision Research Consortium receive approximately $3.2 million (direct costs) of research funds, and $1.8 million of institutional support, annually. The Vision Research Consortium has significant strength in the areas of Visual Neuroscience, Molecular Genetics, Visual System Development, Multi-Center Clinical Trials and Clinical Research. Support is requested for 4 modules which reflect the breadth of these interests: an imaging Module, a Tissue Culture Module, a Biostatistics Module and a Computer Software and Hardware Support Module. These will provide research opportunities not available to individual faculty members. Core support will encourage inter-departmental collaboration, especially between basic and clinical scientists, and support pilot projects leading to new research programs. With the continued support of the Vision Core Grant, a further period of growth and innovative research is expected for the Vision Research Consortium.

SUBPROJECT ABSTRACT NOT AVAILABLE

DESCRIPTION (provided by applicant): In many brain regions, electrical coupling, mediated primarily by Cx36 gap junctions, contributes to neuronal plasticity. In the retina, gap junctions are particularly abundant and they play a key role in the switch from rod to cone pathways. This is an important example of neuronal plasticity that makes the retina a useful system to study gap junction modulation. The mammalian retina detects light over an enormous range of intensities, approximately 12 log units. Furthermore, there are distinct pathways through the retina for both rod and cone signals, in part mediated by gap junction connections. The goal of this research is to test rod and cone connections and determine the physiological role of gap junctions in rod and cone pathways. Specific Aim1 is to map the connections of rods and cones to bipolar cells. By filling single rod bipolar cells in rabbit, we can determine their rod or cone contacts unambiguously. Most ON cone bipolar cells can be labeled via their coupling with AII amacrine cells, providing a novel way to view ON bipolar connections with rods and cones. Controversial results from mouse retina suggested two types of rod bipolar cell (RBC), one of which receives cone input. We will test this hypothesis by recording from mouse bipolar cells and correlating physiology with confocal analysis of rod/cone contacts. In Specific Aim 2, the working hypothesis is that there are 3 kinds of photoreceptor coupling, cone/cone, rod/cone and rod/rod, all using Cx36. Taking a transgenic approach, using rod-specific and cone-specific Cx36 KO mice, we will correlate single photon responses in rods with Neurobiotin coupling patterns and the distribution of Cx36 gap junctions. This will identify the rod connexin and establish the physiological role of Cx36 in rod/rod and rod/cone coupling. Specific Aim 3: AII amacrine cells are a well-known example of well-coupled network. Plasticity in this network is responsible for major changes in retinal circuitry underlying the switch from rod to cone pathways. There is circumstantial evidence that dopamine modulates AII coupling but no direct physiological evidence. We have developed a novel technique in mouse retinal slices to estimate AII network coupling. We have validated this approach using gap junction antagonists and Cx36 KO mice. Here, we report for the first time that dopamine modulates electrical coupling in the AII network. The mammalian retina is a self-optimizing network of around seventy different neuronal cell types. The retina detects light but in addition it is continually adjusting to the intensity and pattern of the visual scene to provide the best output. It is important to understand the structure and function of retinal circuits. Such knowledge will be required to test if transplanted or regenerated cells form appropriate functional connections.

ABSTRACT NOT PROVIDED

PROJECT SUMMARY The Houston Area Vision Training Program is a long-standing collaborative effort among 38 experienced Vision Research faculty at the University of Texas Health Science Center (UT) and the University of Houston College of Optometry (UHCO). The primary goal of the Program is to provide high quality training in well-equipped labs by preceptors with strong records of NEI funding, peer-reviewed publication, innovative research and previous training experience. A specific training program is outlined which provides laboratory training in either basic or clinical research with strong components of relevant course work and a rich environment of seminars and visiting scholars. The co-PIs will be Dr. Massey from UT and Dr. Frishman from UHCO. There are 19 investigators at each institution who together hold 21 R01s and 2 R21s from NEI. In addition, both UT and UHCO hold Vision Core Grants. Support is requested for 4 predoctoral positions and 2 post-doctoral positions. This includes 1 new postdoctoral position to support those clinicians with OD degrees who choose to enter the PhD program. In the last funding period, this program supported 15 Ph.D. candidates including 12 predoctoral students and 3 ODs who entered the PhD program. Of these 15 trainees, 10 completed their PhDs , 4 are close to completion and 1 obtained a MS degree. Of the 10 trainees who graduated with PhDs, 9 have research or academic positions. One had obtained an F31 during training, one was awarded an NEI R01 after training and 1 has a tenure-track position. Of the 4 postdoctoral trainees, 3 were ODs in the PhD program and one was a recent PhD. They are all research faculty and/or still in training. Two of the 3 ODs obtained K-Awards during training and the most recent postdoctoral trainee has a pending F32 Award. From a public health perspective, it is important to train the next generation of Vision Researchers. As life expectancy is prolonged, visual problems will be more frequent and will require new methods of treatment. This will require a multidisciplinary approach by researchers specifically trained in Visual Science to apply the latest technology to problems of the Visual System.

DESCRIPTION (provided by applicant): This is an application by a group of 10 investigators to install a Zeiss LSM 780, a new generation confocal microscope suitable for multi-channel analysis of retinal circuitry and development. This model has pig-tail laser inputs to accommodate a variety of laser lines, high efficiency/low noise detectors and spectral analysis. These developments will make it possible to conduct 4 or 5 channel labeling experiments. The LSM 780 is self contained and menu-driven making it suitable for a multi-user facility. The instrument will be installed in the Confocal Module of the Vision Core Grant housed in the Department of Ophthalmology. This Vision Core facility is heavily used by 10 investigators conducting research on the visual system, including 8 supported by NEI, NIH programs. The service contract will be supported by the Ophthalmology department and there will be no usage charges for this group of investigators. The group is very well known for imaging studies of the retina with 6 recent cover pictures in peer-reviewed journals. In the broadest terms, we seek to understand the structure, function and development of the mammalian retina; such knowledge may lead to new strategies to alleviate visual disorders resulting from photoreceptor degeneration, glaucoma or diabetic retinopathy. PUBLIC HEALTH RELEVANCE: The mammalian retina is our window to the visual world. It is a complex self-optimizing network built from approximately 60 types of neurons. We will use multi-channel imaging techniques to discover new cell types, to relate structure to function, to decode neural circuits and to follow the steps of retinal development. A sound base of such knowledge is essential to develop new strategies to alleviate retinal conditions leading to blindness.

IMAGING MODULE ABSTRACT The purpose of the Imaging Module is to provide instrumentation and training for high-resolution, multi-channel imaging and quantitative image analysis to the members of the Vision Core Group. We strive to obtain the latest technology for our confocal facility, which can support up to 5-channel labeling in a centrally located core facility accessible to all Core Grant members. In the confocal facility, we will have three Zeiss confocal microscopes, (Zeiss LSM 510 META, Zeiss LSM 780 and Zeiss LSM 800 with Airyscan (on order)) plus a Zeiss Apotome 2 mounted on an Axio Imager. After training, authorized users may sign up using the on-line calendar on the Vision Core Grant web page, which also contains instructions and a primer on multi-channel confocal microscopy. A Phoenix Research OCT system is available for small animal work. An initiative to provide 2-photon imaging has been started with a commitment of support and a goal to make this available during the first year of the program. Technical assistance and training for confocal microscopy, OCT and image analysis are available from a microscopist and software specialist supported by the Module. Programs for off-line image analysis (ZenLite, and ImageJ) are widely distributed throughout the user group and the Module holds licenses for several additional programs (ZenBlue, Imaris and Image Pro). The Module will also provide access to a comprehensive library of fluorescent secondary antibodies for multi-channel imaging. Priority use is assigned to NEI supported research and pilot projects in vision research. Because the Imaging Module will support the service contracts on the microscopes, confocal microscopy will be freely available to all members of the Vision Core Grant at no charge. We believe this is a key factor in the careful analysis of confocal material. The no-charge policy actively encourages new users and junior faculty because it allows the time to learn about confocal imaging.

ABSTRACT The Vision Research Group at The University of Texas in Houston (UT) has significant strength in the areas of Visual Neuroscience, Molecular Genetics, and Visual System Development. Core Grant support is requested for an Administrative Module and 3 service Modules, which reflect the breadth of these interests: an Imaging Module, a Molecular Resources and Services Module, and a Biostatistics and Computation Module. These Modules will provide expertise, research opportunities and infrastructure beyond the reach of individual faculty members. Core Grant support will encourage collaboration and support pilot projects leading to new research programs. Vision Research has been identified as an area for investment and development at UT and we provide documented evidence of institutional commitment to support our research effort. Specifically, the administration has provided funds to purchase a new confocal microscope (Zeiss LSM800 with Airyscan) to upgrade the Vision Core Imaging Module. Thus, significant resources, personnel and laboratory space have been committed to the Vision Research Group, a faculty group that crosses departmental lines. The Vision Research Group currently holds 8 qualifying NEI-supported R01s. In the last year, we recruited 2 new faculty members. Our new recruits were attracted, in part, by the outstanding facilities supported by the Vision Core Grant. In total, the Vision Research Group now consists of 15 vision scientists from the McGovern Medical School, the School of Public Health and the UT M.D. Anderson Cancer Center. The members of the Vision Research Group receive approximately $4.4M (direct costs) of research funds annually. In 2015/16, the Vision Research Group received almost $3.8M in institutional support for salaries, new faculty and equipment. With the continued support of the Vision Core Grant, a further period of growth and innovative research may be expected for the Vision Research Group at UT.

ADMINISTRATIVE CORE ABSTRACT The UT Vision Core Grant is divided into an Administrative Module and three service Modules: the Imaging Module, the Molecular Resources and Services Module, and the Biostatistics and Computation Module. The purpose of the Administrative Core is to provide administrative support and oversight of the Vision Core Grant. This will include professional grant management, planning, meeting preparation and purchasing. Scheduling for Vision Core Grant resources such as microscopes will be arranged by on-line calendar. The allocation of Vision Core Grant services, such as Tissue Culture or Computer Programming and Biostatistics, will be scheduled via the Module Director according to a formula designed to reserve some open time for easy access. Oversight of Core Grant resources will be performed by Module Directors reporting to the Program Director and the Executive Committee. An open General Meeting may also make recommendations to the Executive Committee. In turn, the Executive Committee will be overseen by the Advisory Committee, which includes independent members from outside the Vision Core Grant faculty. Module evaluation will use the following metrics: usage, number of projects, number of members served, papers published, and grants submitted. In this way, we will ensure the equitable distribution of resources and provide open access to all.

Intrinsically photosensitive retinal ganglion cells (ipRGCs) play a key role in transmitting non-image-forming visual information to the brain. Recent evidence has implicated ipRGCs in conscious vision as well as in serious conditions such as migraine pain and seasonal affective disorder. Despite the fundamental importance of ipRGCs in the visual process, the underlying synaptic mechanisms and circuits that control ipRGC function are unknown. IpRGCs express their own photopigment - melanopsin and, at high light intensities, intrinsic responses drive ipRGC function. However, surprisingly, at lower intensities, even in the photopic range, ipRGCs are predominantly driven by rods and not cones. These data suggest that a sustained signal originating from rods must travel through the retina to carry information about irradiance to ipRGCs. In this proposal, we will test the primary hypothesis that the irradiance pathway through the mammalian retina is driven via rod-to-cone gap junctions. Our preliminary studies provide evidence that a novel irradiance pathway contains the following elements: rod?rod/cone gap junction?cone?ON cone bipolar cell?ectopic synapse?M1-type ipRGCs and dopaminergic amacrine cells (DACs). In turn, M1 ipRGCs drive non-image-forming visual behavior such as the pupillary light reflex and circadian photoentrainment, while dopamine release may control network adaptation in the retina. To test these hypotheses, we have developed and validated several mouse lines in which Cx36 has been conditionally deleted in either rods or cones, and therefore lack rod/cone gap junctions. In Aim 1, we will test the hypothesis that rod/cone gap junctions are required to drive the PLR, circadian photoentrainment and negative masking, non-imaging-forming visual functions also driven by M1 ipRGCs. In Aim 2, we will test the hypothesis that rod/cone gap junctions are also essential for the release of dopamine, in the mammalian retina. Furthermore, we will test the hypothesis that dopamine-dependent network adaptation relies on the irradiance pathway via rod/cone gap junctions. In Aim 3, we will test the function of the irradiance pathway at two key points: rod/cone gap junctions and ectopic bipolar synapses in the inner plexiform layer. In summary, we propose that rod/cone coupling generates an irradiance signal transmitted via ipRGCs that not only controls the pupillary light reflex, it also entrains the circadian clock every day. The biological influence of the circadian clock is pervasive yet it may be driven via gap junctions between the first two cell types in the visual system. Furthermore, there is a link between dopamine and myopia. If, in turn, the irradiance pathway controls dopamine release, this may inform a new approach to myopia.

SUMMARY Electrical synapses, also known as gap junctions, occur frequently in all nervous systems, including the human brain. They are composed of connexins, arranged to form intercellular channels between adjacent, coupled cells. Connexin36 (Cx36) is the predominant connexin in the CNS. In many brain and retinal circuits, gap junctions provide direct and specific connections between cells. In addition, electrical synapses mediate network properties such as signal averaging, noise reduction and synchronization. However, because of their small size, gap junctions are not visible in large-scale serial EM data sets. For these reasons, gap junctions tend to be under-reported or simply ignored. The objective of this proposal is to develop a combined approach to image gap junction connectivity in EM datasets and, in addition, to estimate the size, strength, and plasticity of gap junctions. We will study regions of the retina that contain gap junctions of dramatically different sizes and shapes, to allow us to correlate structure and function. Aim 1 will use high-resolution confocal microscopy to determine connexon number at large and small gap junctions. Analyses will determine the number of connexons per gap junction. These methods will provide a general-purpose tool to determine the size of gap junctions for use in all brain regions. Aim 2 will use 3D-EM imaging to allow unambiguous identification of gap junctions in FIB-SEM images, which will follow with first-ever immunogold quantification of a membrane-bound protein in 3D-EM structures. These studies will allow high-resolution quantification of gap junctions and proteins in identified neurons. Aim 3 will use electrophysiological measures to determine coupling conductance and then develop models to calculate the maximal potential coupling conductance from the morphological data by multiplying the number of channels/gap junction [Specific Aim 1] times the connectivity (the number of gap junctions between coupled cells) [Specific Aim 2], times the unitary conductance of Cx36. Using paired recordings, we will obtain direct physiological measures of the junctional conductance between coupled cells. Then, by comparison with the potential maximum calculated from the morphological data, we can calculate the open channel probability and place realistic limits on the operating range. These are the fundamental properties required to understand the function of gap junctions in neuronal microcircuits. This program is an exact match for one of the listed areas, ?Tools to identify gap junctions and characterize electrical synapses? in the Funding Opportunity Announcement, RFA-MH-20-135.