Viktor N. Kharazia, Ph.D. - US grants

DESCRIPTION (provided by applicant): The Histology and Microscopy Core facility at the Ernest Gallo Clinic and Research Center (EGCRC) at the University of California, San Francisco provides shared equipment and training in methods that use microscopy for investigating molecular and structural changes in the brain that underlie drug and alcohol dependence. These questions have been traditionally addressed using immunocytochemistry and in situ hybridization, and for the past decade, with the NIH-developed method of laser capture microdissection (LCM), which offers the possibility of selectively collecting specific cell populations from counterstained histological sections. The original infrared (IR) laser-based PixCell IIe system (Arcturus) was purchased by the EGCRC in 2001. Since then this instrument has allowed our investigators to measure mRNA abundance in subpopulations of invertebrate and mammalian cells by RT-PCR and microarray technology. However, over the last several years, technical drawbacks with this IR laser-based LCM design have become evident. First, it can optimally use only 5-8 micron thick sections and is thus limited to analysis of small amounts of tissue. Second, our PixCell IIe system is manual and cannot outline and then dissect out a brain region using an automated function. Furthermore, to obtain the most effective IR pulses, one must refocus the manual laser before and during tissue collection, which is very time-consuming. In addition, investigating regions of the brain infected with viruses for RNA interference studies has proven very difficult to perform due to the need to collect many thousands of cells from each animal. Therefore, despite a significant need for LCM in our experiments, our current system is extremely inadequate. Recently, LCM vendors have developed systems that use ultraviolet (UV) lasers as a cutting tool and this development has proven to be a major improvement. Since our PixCell IIe is not upgradeable, we now seek to acquire the most up-to-date instrument, an LDM7000 from Leica Microsystems. The requested instrument will be equipped with the highly effective UV cutting laser, automated region collection and other options that will greatly assist in obtaining better samples at reduced cost and time. With the LDM7000 our investigators will be able to continue to perform NIH-funded experiments and avoid the serious limitations imposed by the outdated equipment currently at the EGCRC.

The Vector and Imaging Core (Core Component 3) will provide centralized services, supplies, and shared equipment to all Research Components and Pilot Projects that propose use of viral vectors for gene silencing by RNA interference in select brain regions. Core personnel will design effective shRNA molecules, produce high titer viruses, and perform in vitro and in vivo testing. Core personnel will also produce viruses for overexpression of genes of interest. The Vector and Imaging Core will continue to provide a range of histological services for the Research Components and Pilot Projects, by providing feedback on stereotaxic coordinates of viral injections, volume of infections, and types of infected cells. The Core will analyze expression of targeted genes by laser capture microdissection combined with quantitative PCR mRNA analysis, and by immunocytochemistry. Core personnel will continue to perform experiments on the cellular localization of signaling proteins, and will further extend detection capabilities by performing high resolution in situ hybridization and immunocytochemistry for signaling proteins in dendrites. Additional Core responsibilities include: oversight of digital stereotaxic equipment, imaging equipment; training in stereotaxic surgical techniques, histology, imaging and image processing; supervising students and postdoctoral trainees; and generating standard procedures for techniques used by the Core. By housing vector and imaging services in one core, efficiency and cost can be optimized since core personnel can focus their skills on techniques that are labor intensive and universal to several research projects. This will allow investigators of Research Components and Pilot Projects to focus on scientific questions involved in studies of specific candidate proteins, while avoiding technical errors and inconsistency in application of methods that the Vector and Imaging Core can perform to manipulate expression of these proteins.