Core Director: Catia Sternini, M.D., Professor of Medicine and Neurobiology, UCLA School of Medicine

Associate Directors: Nicholas Brecha, Ph.D., Professor of Neurobiology and Medicine, UCLA School of Medicine, Nora Rozengurt, Ph.D., Departments of Pathology and Laboratory Medicine

Research Associate: Steven Young, Ph.D.

OBJECTIVES

The overall goal of the Morphology/Imaging Core is to enhance the effectiveness of the CURE center program by providing facilities, training and assistance for morphological, imaging and functional studies of transmitters, receptors, channels and other integral proteins in native and transfected cells, to CURE investigators with independently-funded research projects.

CORE SERVICES

A. Immunohistochemistry for 1) the localization of transmitters, hormones, receptors, growth factors and transporters using single and multiple labeling and 2) the investigation of receptor trafficking following ligand stimulation by means of visualizing ligand-induced receptor internalization, intracellular sorting and recycling in transfected and native cells, which can be achieved by combining immunohistochemistry with confocal microscopy.

B. Assistance and training for Light Microscopy and Digital Photography.

C. Computer assisted image processing and analysis for quantitative determinations of phenotypic changes in gene expression, for morphometric analysis of different cell populations (including measuring areas, or length or surface of villi, counting specific histochemically or neurochemically identified cell populations), and for the quantification of receptors at the plasma membrane and intracytoplasmic compartment;

*D. Confocal microscopy for 1) the analysis of intracellular pathways involved in cellular signaling, 2) the cellular and subcellular localization of proteins, and 3) the definition of the spatial relationship of receptors with their ligands and with neurochemically-defined and distinct populations of neurons and endocrine cells. The confocal facility has been upgraded by the addition of a newly acquired Zeiss LSM 510 Meta equipped with Axioplan 2 microscope, which is located within the Core facilities in the main UCLA campus and is equipped for live and fixed cell imaging. The 510 Meta is a state-of-the-art confocal microscope with highly efficient, affinity corrected optics, multi-channel detectors, emission fingerprinting, multiple pinholes, and user-friendly software with wide range of functions including two separate 3-D reconstruction options. The Meta feature places this system at the cutting edge of confocal microscopy; with the ability to collect images representing emissions from 440nm to 785nm in 11 nm sequential bands, the Meta will provide for accurate separation of the signals from multiple (4-6) fluorophores from a single preparation. A Zeiss LSM 410 equipped with Ar/Kr and UV lasers connected to a Zeiss Axiovert 135 will also continue to be available for fixed tissue analysis in the Department of Neurobiology.

E. Ca2+ imaging in conjunction with imaging GFPs and other intracellular fluorescent probes for the analysis of signal transduction pathways in single cells. The Ca2+ imaging system available is a Zeiss AttoFluor Ratio vision with Axiovert microscope, quartz optics and a temperature control perfusion chamber with an Eppendorf microinjection system to introduce non-permeant dyes into cells for the visualization of intracellular events. This approach will allow for quantitation of receptor levels in individual cells and assessment of purity of cell preparations.

F. Fluorescent imaging system for in vitro measurement of intracellular cAMP levels using single excitation dual emission configuration in single cells with attached microinjection system to introduce non-permeant dyes and non-permeant inhibitors or facilitators of signal transduction pathways into cells for the direct visualization of intracellular events is available in conjunction with the fluorescent imaging systems.

*G. Animal pathology service will provide a detailed anatomical analysis of possible phenotypical or pathological alterations associated to genetic manipulations and a comprehensive and systematic characterization of novel genetically engineered mouse models. This service includes different methods of tissue procurement and fixation, high quality routine tissue processing, embedding, cutting, staining, electron microscopy, morphometry, and pathological characterization of the animal models carried out by an experienced veterinary pathologist.

*H. Access to facilities for non-invasive neuroimaging approaches for investigating the anatomical and biochemical-physiological functions of the brain with high anatomical and temporal resolution as well as to computational infrastructure for archiving imaging will be provided through the Core. Functional MRI is capable of observing the response of cerebral perfusion to visceral and cognitive stimuli. PET and SPECT are capable of assessing a wide variety of cerebral physiological functions, including perfusion, glucose metabolism, neuroreceptor density and neurotransmitter release. Perfusion and metabolism abnormalities have been observed in stress-related illnesses. For instance, abnormalities in the opioid system are seen in patients with neurovisceral and chronic pain syndromes. Electrophysiological imaging has several unique features that complement the other modalities. It observes neuronal functional rather than metabolic correlates of function. It is recorded on a physiological time scale, providing a temporal dimension in assessing physiological function; i.e. are responses abnormally slow or fast. It is also entirely non-invasive, portable and inexpensive. Computational infrastructure for archiving imaging will be available to CURE: DDRCC investigators. Software packages for image analysis and modeling will be licensed and supported for investigators. These include MEDX, SPM, MATLAB, IDL, MRICRO, IMAGEJ, SPSS, SAS. Consultants will be available to help investigators with the use of these packages.

Teaching contemporary morphological and cell imaging methodologies, histological skills and techniques, and the application and use of non-invasive human imaging approaches, as well as appropriate controls and common methodological pitfalls, are a high priority service offered by the Core with the intent of training single investigators, their technical staff and fellows. Core personnel, including the Director, Co-Director and Associate Directors, meet with investigators who are interested in using the experimental approaches offered by the Core and discuss the projects to be performed and the specific experimental approaches and techniques. Given the types of services offered by the Core, which are labor intensive and require consultation with the Core Directors in addition to the staff personnel, the major functions of the Core are (1) to teach and train investigators or their co-workers in the performance of specific techniques and experimental approaches including the use of the appropriate equipment for the planned project, (2) to make facilities and equipment available for investigators once they have learned specific techniques, and (3) to facilitate access to equipment available in the institution (VA or UCLA campus). Individual investigators provide animals and cover the cost of specific reagents and supplies needed for their projects. General supplies, such as basic buffers and chemicals, glassware, and plastic ware are available for investigators in the Core facility.

An important feature of this Core has been and will continue to be a close interactive relationship among Core users and personnel. Frequent discussion and demonstration during the initial training period are provided in the performance of experimental procedures and in the evaluation and interpretation of experimental results. Once trained, investigators have access to the equipment available in the Core laboratories to pursue their studies on their own, without direct participation of the Core personnel, except when requested for consultation.

BACK