RCMI Research Activities

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ADMINISTRATIVE COMPONENT

  • Administrative Leadership Core – Dr. Eddy Ríos-Olivares

The Administrative Leadership Core (ALC) of this application has been designated to assume the leadership in developing an adequate research environment to conduct the Universidad Central del Caribe to a more productive scientific enterprise. This setting encompasses a Leadership Core with experienced administrators and staff in research management, a Scientific Research Development Unit to foster research faculty development in basic and clinical investigation, and a Data Management and Statistical Research Support unit as a complementary specialized service to the research efforts by the faculty. The ALC will act as the flagship of the overall proposed plan and will be accountable for its execution. The research plan that this administrative component proposes to oversee, contemplates to strengthen the Retrovirus Research Center by providing expert personnel and the HIV and Substance of Abuse Laboratory Core; to strengthen the Neuroscience Research Center, which will nurture an intellectually stimulating environment (mentoring, collaborations, exchange program, a Behavioral Testing Facility and a Neuronal and Glial Culture Facility) for neuroscience research, and create a Cancer Research Center, which will have a cluster of investigators studying dietary compounds as potential cancer preventive and therapeutic agents. In addition, the ALC will continue supporting essential research cores (Optical Imagine, Protein and Nucleic Acid Core Facilities and Common Instrumentation Area and Services); and create a Biomedical Proteomics Facility. Furthermore, the ALC will provide support to five pilot projects in the areas of retrovirology, neuroscience and cancer and implement an RCMI evaluation plan to appraise accomplishments. The judicious implementation of this plan is considered pivotal to consolidate UCC’s past, present and future research efforts.

 

  • Scientific Resources Development Unit – Dr. Luis A. Cubano

The GOAL of the Unit is to increase UCC’s capacity to perform world-class research and attract first-rate researchers. The SPECIFIC AIMS of the SRDU for this cycle of support are: 1) Increase the critical mass of researchers at UCC involved in translational research; 2) Increase the number of translational research projects performed at UCC; 3) Provide support to investigators in pre- and post-award management; 4) Increase the number of publications in high-impact peer-reviewed journals; 5) Increase interdisciplinary inter-institutional research collaborations; 6) Increase the Institution’s capabilities to obtain non-minority external funding; 7) Increase the amount of funding provided by non-federal sources of funding. The goal of fostering translational research at UCC with a focus on HIV/AIDS, neurological disorders and cancer is directly relevant to human health and the elimination of health disparities.

 

  • Data Management and Statistical Research Support Unit – Dra. Diana Fernández

The overall goal of the DMSRSU is to become the data management and statistical research support Unit for RCMI and other UCC researchers in order to enhance the level of scientific research excellence in the institution. In plus, the DMSRSU will continue to house and maintain the HIV data bank in coordination with the Retrovirus Research Center. For the renewal, the DMSRSU has the following objectives: (1) Consolidate the infrastructure of the DMSRSU as an independent unit; (2) Provide support for study design and statistical analysis to the multiple funded and unfunded studies of RCMI and other UCC researchers; (3) Provide data management and quality assurance research support to the multiple funded and unfunded studies of RCMI and other UCC researchers; (4) Continue the study of the natural history of HIV disease in coordination with the RRC; and (5) Contribute to the research development career of faculty in coordination with the Scientific Human Resources Development Unit (SHRDU) and the Retrovirus Research Center (RRC). This unit has a strong infrastructure which includes the following subunits: Data Abstraction and Management; Data Entry; Quality Control; Data Analysis and Consultant; and Administrative and Computer Systems. Each of these subunits consists of experienced professionals readily available to assist researchers and to provide data management and statistical research support to investigators.

 

RESEARCH CENTERS

Retrovirus Research Center – Dr. Robert Hunter

The Retrovirus Research Center (RRC) is a facility that promote and stimulates research addressing the HIV and AIDS epidemic in Puerto Rico. The RRC is composed of a Leadership Core and a Laboratory Core. In addition it works intimately with the Data Management and Statistical Research Support Unit (DMSRSU), which manages a longitudinal database of over 4,000 patients with HIV infection. It’s considered unique in Puerto Rico since it is the only one in which full integration of the research effort with the health care delivery facility is in place. The aim of the RRC is to promote the study of HIV- infection as a multidisciplinary research arena in which the clinical, immunological, virological, psychosocial and behavioral parameters need to be integrated into a coherent research strategy. The center agglutinates multidisciplinary researchers interesting in describing and understanding key elements of HIV infection according to an ecological view. The RRC has an important role in continuing the research agenda with this patient population with the goal of diminishing the spreading nature of the HIV epidemic in the Island. The Leadership Core coordinates and facilitates an intellectually stimulating environment for HIV/AIDS research by playing a proactive role in generating hypothesis-driven studies. This multidisciplinary vision of the RRC is responsive to the vision of the National Institutes of Health their role of studying the complexity of diseases. Our objectives for the next five years will include 1. Provide the support for the study of the multidimensional aspects of HIV infection, by planning and stimulating HIV/AIDS research, scientific productivity and hypothesis driven studies. 2. Provide support for HIV research activities. The resources include the HIV Data Registry, the specialized laboratory and the plasma and lymphocyte repository. 3. Contribute to the research development career of the RRC faculty members and research staff in coordination with the DMSRSU and the SRDU. 4. Continue to provide support for community involvement in research activities. 5. Develop strategies to become a RCMI independent HIV Research Center.

 

  • HIV and Substance of Abuse Laboratory Facility – Dr. Eddy Ríos-Olivares

The HIV and Substance of Abuse Laboratory Core (H-SALC) will continue providing human and physical resources needed to support studies in the areas of HIV/AIDS and related diseases as well as in substances of abuse. This support is extended to the Retrovirus Research Center, Center for Addiction Study, Comprehensive Center for the Study of HIV Disparities and other centers of the RCMI Program. In addition, it will continue with the existing collaborative activities with at least three communities and government based agencies. Furthermore, it will provide administrative and scientific support to pilot projects with the participation of consultants and mentors. Therefore, the research plan for the Renewal Proposal is directed toward: 1) Providing a centralized, well-structured laboratory supportive of research activities undertaken by centers and programs, including those handling infectious material and immunodeficiency diseases (HIV/AIDS, HCV, cancer, and vaccine development). 2) Improving and introducing new methodology to test the effect of addiction-related drugs (cocaine, heroin, methamphetamine and, methadone, and buprenorphine) on the immune system, the glial-neuro-network and HIV (i.e., NeuroAIDS), and HCV infectivity. 3) Upgrading and replacing obsolete instrumentation and acquiring new equipment to meet the increasing need of research projects for redirecting their methodology and making it more amenable to the translational research trend, 4) Strengthening existing and new affiliations with community service agencies to facilitate the involvement of specific disease populations in our research and services efforts, 5) Providing scientific and administrative support to pilot projects with the participation of consultants and mentors.

 

  • Cancer Research Unit – Dr. Robert Hunter

The objective of the proposed Cancer Research Center is to build cancer research capacity at UCC and increase minority participation in cancer research by training the next generation of minority cancer researchers. To achieve this objective, the following Specific Aims will be carried out: 1) Expand the infrastructure to conduct cancer research at UCC; 2) Increase the number of cancer researchers at UCC; 3) Develop translational and clinical cancer research at UCC; 4) Establish a seminar series on cancer research and continuation of the cancer group meetings and journal club; 5) Educate the community about cancer prevention/early testing; 6) Support a Pilot Project from a junior faculty member.

The center will be led by a senior researcher and will be organized into three main areas: 1) Testing the efficacy of novel cancer therapeutics in cell and mouse models, 2) genomic and protein array analysis of cancer pathology, 3) establishment of a cancer tissue repository from patients of mainly Hispanic origin. The goal of the Cancer Research Center is to facilitate translational research in the prevention and treatment of cancer.

 

  • Neuroscience Research Center – Dr. Misty Eaton

The primary mission of the Neuroscience Research Center is to foster and conduct science of the highest caliber that advances the understanding of brain function and diseases that affect the nervous system. The Center maintains a quality research environment highly conducive to productive and clinically-oriented basic research focused on Neuroscience. The Center’s scientists share common interests in understanding the biology of neurological disorders and work toward the shared goal of ultimately developing novel methods of diagnosis and treatment of the disorders.

The major goals of the Center are: Our long term goal is to support a scalable and sustainable group of neuroscience researchers who are focused to determine mechanisms causing, and to discover novel strategies to prevent and treat neurological disorders. This will be accomplished by facilitating dialogue between basic and clinical scientists to promote development of collaborative and translational research in the field of neuroscience.

 

  • Behavioral Testing Facility – Dr. Legier Rojas

The main goal of the Behavioral Testing Facility (BTF) is to cover a need associated with the development of the basic sciences and clinical community at the Universidad Central del Caribe (UCC). Additionally, BTF has extended its services to all Puerto Rico’s entire scientific community. We are proposing five specific aims for the renewal proposal.

Specific Aim 1. To foster the usage of BTF by basic scientists and clinicians in Puerto Rico. Enhancement in the use of the facility will be addressed by the integration of two additional functional units within the BTF core: the Human Behavioral Phenotyping Unit (HBPU) and the Animal Behavioral Phenotyping Unit (ABPU). Specific Aim 2. To continue providing highly productive working conditions for the use of the existing equipment and the environment necessary for both ongoing and new projects oriented to translational research. Specific Aim 3. To continue the training of research assistants and other personnel and BTF’s users in methods, equipment, experimental protocols and software used in behavioral experiments. This training will include new internet based methodologies to provide even greater access to our facilities. Specific Aim 4. To intensify the collaborative research at the UCC community, and between UCC and other local and Continental USA Universities, by effectively marketing the facility through the cooperative research network “Research Centers in Minority Institutions Translational Research Network” (http://www.rtrn.net) which is facilitating the translational research in health disparity areas. Specific Aim 5. To help the scientific community involved in experiments to acquire data and visualize animal behavior and/or psychomotor alteration related to translational research by maintaining updated the analytical methodologies. Our Behavioral Testing Facility has served > 30 researchers, and currently 10 investigators are using the facility. Four new proposals recently submitted to federal agencies are including usage of BTF in their experiments.

 

  • Neuronal and Glial Culture Facility – Dr. Pedro Ferchmin

The Neuronal and Glial Culture Facility or NGF is a core facility structured to provide cultures of interest to neuroscientists. In some cases, the NGF provides training and starting supplies to investigators that prefer to learn the procedures and then continue doing the cultures themselves. In each case, the NGF facilitates and speeds up research. The NGF does not occupy physical space, it uses the equipment of the central cell culture facility of the RCMI and the NGF technician uses the equipment and space of a laboratory in the Department of Biochemistry. Therefore, the NGF is based on a space and equipment-sharing basis. This core facility consists of a coordinator (PAF) and a technician, Mrs. Brenda Cuadrado (BC), experienced in neuronal cell culture. Among other skills, she is able to prepare neuronal cultures from fetal cortex or hippocampus, astrocytes from neonatal cerebral cortex, organotypic hippocampal slices using the Stoppini method. BC was trained at the UCC (by PAF), by Dr. C. Ghiani from UCLA and BC traveled to UK, Lexington (USA), to learn organotypic slice culture in the laboratory of Dr. Littletone. During the last year, Mrs. Cuadrado has learned immunocytochemical methods under the guide of Dr. H. Yeh (consultant), the NGF coordinator (PAF) and Dr. H. Martins. The immunocytochemical methods are used to monitor the cultures provided by the NGF. By dedicating one technician to produce a significant part of the cultures done at the UCC alleviates in part the crowding in the culture room, the duplication of equipment and saves sera and reagents. As result of this facility, the investigators no longer need to develop “from scratch” the cultures. This core facility is based on a similar arrangement in the laboratory of Dr. de Vellis’ laboratory (UCLA) where a single person is dedicated to provide cultures for all the members of this prestigious group. The proposed budget consists of supplies and personnel. Salaries of the technician (BC) the coordinator (PAF) and the consultant costs are included. Supplies include media, sera, antibodies for quality control and further development of cultures. The objective of this unit is not to exclude anyone from using the existing cell culture facility but by providing a better alternative discourage the initiation of individual neuronal culture laboratories throughout the UCC. By doing so, we will increase the efficient use of space and equipment. By providing the expertise in brain cell culture and the labor involved, this core facility will leave more time to the investigators for the scientific endeavor.

 

RESEARCH CORES

  • Optical Imaging Facility – Dr. Priscila Sanabria

The Optical Imaging Facility (OIF) is core facility established to provide the equipment, training, technical and scientific expertise necessary to optimally carry on applications requiring image capture and processing from live and fixed specimens. The facility houses widefield fluorescence microscope imaging systems, a laser confocal microscope and other standard equipments for sample preparation and live cell culture and maintenance. The OIF also provide services for the preparation of specimens through its Immunocytochemistry Laboratory and technical support for each of the components. Our continuation goal is to support and help our faculty to identify and develop relevant research that would address significant health issues in our local community. In order to accomplish the OIF mission, our specific aims are:

  • To provide technical support to optimally perform studies that requires immunocytochemistry and quantitative fluorescence imaging capture and analysis.
  • To provide instrument access and training on the effective utilization of microscope-based equipment to carry on applications that rely on image capture and processing from live and fixed specimens.
  • To disseminate through the academic community in Puerto Rico the capabilities of the OIF and the achievements of its users through the offering of seminars, workshops on imaging technology and maintaining a website that includes information about the facility operation, instrumentation reservation and other educational resources.
  • To enhance UCC’s scientific environment by providing young faculty and graduate students state of the art imaging technology to obtain preliminary data to apply for external funding and helping established investigators to innovate their research and increase their publication record.
  • Protein and Nucleic Acid Core Facility – Dr. Krishna Baksi

The Protein and Nucleic Acid Core Facility (PNACF) will aid the UCC investigators in the transition from the more classical studies to a more molecular and up-to-date analysis of the problems being addressed by each investigators. PNACF will focus on faculty training, seminars, training on specialized molecular biology techniques, protein expression and protein purification and characterization. Therefore, the PNACF continues to be an important core facility for the maintenance and future development of research at this institution. Specific Aims are:
(1) To continue helping researchers with training/service current molecular biology techniques and training other researchers including post-doctoral fellows and research assistants, on the use and maintenance of specialized equipments.
(2) To introduce two powerful techniques, namely gene microarray and nanotechnology in collaboration with the core facilities of other institutions in the mainland USA.
(3) To sponsor ‘hand-on” workshops in molecular biological techniques, cell-free protein expression and fusion protein purification techniques given by the invited experts in each of these fields. This will help the UCC researchers to directly address their research problems and to obtain preliminary data for publications/presentations/external funding.
(4) To continue to be a resource center where the UCC faculty can obtain technical information and cost estimation for molecular biological/protein analysis techniques and services that are not available at UCC.
(5) To continue helping the researchers for the computerized analysis of nucleic acid and protein data.

  • Common Instrumentation Area and Services – Dr. Misty Eaton

The Common Instrumentation Area and Services Unit (CIAS) has the objective of providing the investigators at UCC with the major equipment and services that are needed for developing and executing high quality research projects. There are over 25 very active investigators (many with projects funded by RCMI, SNRP, MBRS, DOD, INBRE and CNS) who rely heavily on this core facility. The CIAS houses instrumentation such as high speed and ultra-centrifuges, a gel documentation system, a scintillation counter, a gamma counter, water distillers and ice machines that are not available elsewhere at the University and therefore, the CIAS fosters equipment sharing and centralizes maintenance of equipment. In addition, the CIAS provides a common cell culture room for new and established investigators who do not have the facilities in their own laboratories and as a backup facility for investigators with cell culture hoods and incubators. Under the umbrella of the CIAS, there is a machine shop and an electronics workshop. These workshops are maintained by two technicians who are dedicated to fulfilling the needs of the scientific community at UCC. In this proposal, we request funding to continue to conserve and upgrade the existing facilities and resources initially funded by RCMI including the common instrumentation, the electronics shop and the machine shop. The following specific aims form the basis of our plan to maintain, fortify and expand our infrastructure for scientific research.
Aim #1: to provide UCC faculty with the necessary equipment to be productive researchers.
Aim #2: to update the existing instrumentation.
Aim #3: to develop an equipment calibration service
Aim #4: to continue the electronics workshop and precision machine/carpentry shop components.
Aim #5: to continue the Facilities Monitoring System (FMS).

  • Biomedical Proteomics Facility – Dr. Nawal Boukli

The Biomedical Proteomics Facility (BPF) at the UCC will provide researchers with the ability to analyze and identify proteins to address basic and translational research questions that could not be previously studied due to the lack of support in proteomics techniques. The scientific support provided by BPF will facilitate the integration of proteomics into basic and translational research projects and submission of new research proposals to granting agencies.
The following SPECIFIC AIMS will be carried out: Specific Aim #1: Provide scientific and technical support to principal investigators within UCC and other universities in Puerto Rico for the accomplishment of research projects.
Specific Aim #2: Attract new scientist to the technique of proteomics and interest them in establishing collaborations exploiting the technologies available at BPF.
Specific Aim #3: Establish collaboration with scientists in the academia and industry of Puerto Rico.
Specific Aim #4: Provide education and training in proteomic methods. The goal of this facility is the application of sensitive and quantitative technologies to relevant biomedical and basic scientific questions. So far the BPF has played an essential role in supplying 2D gels and mass spectrometry preliminary data for several researchers. Ultimately, the BPF will facilitate the establishment of partnerships between the academia and pharmaceutical industry, situating UCC at the vanguard in protein analysis and contributing to the recruitment of new scientist and the establishment of collaborations in and outside of the island.

 

RESEARCH PROJECTS

  • Proteomic Approach for the Identification of PAI-induced PBMC and CD8+ T Cells anti-HIV Mechanism – Dr. Nawal Boukli

It is now accepted that restoration of the immune function using only highly active antiretroviral therapy (HAART) is incomplete. Because of the emergence of drug-resistant strains of HIV-1, therapeutic immunization strategies are needed to reinforce HAART in the treatment of HIV disease.
We have previously observed that a polyantigenic immunomodulator, known as PAI, which consists of a mixture of inactivated influenza and bacterial vaccine, induces MHC non restricted non-cytolytic anti-HIV-1 activity. Based on our preliminary data documenting viral suppression, we propose to test the hypothesis that PAI induced antiviral activity can be differentially determined by a proteomic approach.
To address this hypothesis, our specific aims are:
Specific Aim #1: To determine differential protein expression in intracellular lysates from target T cells treated with supernatant from effector cells (PBMC, CD8+and CD8+ depleted T cells) by performing two-dimensional gel electrophoresis (2D-GE) and image analysis.
Specific Aim #2: To identify and compare differentially expressed proteins from target T cells treated with PAI by performing peptide mass finger printing using mass spectrometry (MS), stable-isotope labeling by amino acids in cell culture (SILAC) analysis and database search.
Specific Aim #3: To validate by Western blots and/or qRT-PCR the identity of differentially expressed proteins identified by mass spectrometry.
Preliminary findings identified PBMC proteins responsive to PAI treatment. Master maps were compared to assess differences in protein expression. This revealed 47 differentially expressed spots in PAI treated PBMC. The altered proteins were analyzed by tandem MS (MS/MS) for protein identification. After querying the MS/MS data against the NCBInr protein database, we have identified 11 differentially expressed protein spots. We believe that the identified proteins will generate a proteomic signature of the PAI-anti HIV-1 response. We will make the proteome reference map of PAI treatment in different cell type with their respective related HIV mechanisms available for others to use for comparative studies.

  • Ex Vivo Effect of Methadone Use and Withdrawal on Immune Function and H IV-1 replication of CD4+ T-Cells from Chronic Methadone Patients Dr. José W. Rodríguez

Drug abuse remains one of the major risk factors for the spread of HIV-1 infection with opiates being a principal drug of concern. Opiates have been shown to be determining factors in the rate of progression of AIDS. The sum of various in vitro, animal-model, and epidemiological studies indicates that opiates have conditionally variable effects on HIV-1 disease progression. Immunovirological and pharmacological variability are two major factors that seem to dictate whether HIV-1 infection will progress more rapidly or more slowly in HIV-1 infected opiate-exposed individuals. According to previous studies, the immunovirological effect of an opiate drug such as methadone is not well characterized in an ex vivo human system. The hypothesis to be tested in this proposal is that ex vivo methadone use and withdrawal will enhance HIV-1 infectivity in CD4+ T-Lymphocytes by altering cytokine/chemokine profile and upregulating HIV-1 co-receptor and mu-opioid receptor expression. The specific aims will analyze various immunovirological determinants of HIV-1 infectivity to evaluate the effects of methadone use and withdrawal. CD4+ T-Lymphocites will be obtained from HIV-1 negative healthy volunteers and chronic (more than one year) methadone patients. Cells will be acutely infected with HIV-1SF2 (X4/R5 dual virus strain) and then assayed to determine how ex vivo methadone exposure (10-7 and 10-6 M) and abrupt withdrawal: (1) affects HIV-1 infectivity (using HIV-1 p24 ELISA): (2) disrupt cytokine/chemokine profile (using cytometric bead array and ELISA); and (3) modulates the gene and protein expression of extracellular and intracellular molecules (using flow cytometry, quantitative RT-PCR, and Western blot). Statistical analysis will be used to determine significant differences between the different experimental conditions. This study will reveal new cellular and molecular mechanisms involved in methadone-mediated IV-1 replication enhancement. This data can be used to design future in vivo longitudinal studies on the immune and viral modulating effects of methadone in HIV-1 infected methadone Patients.

  • Investigation of Reishi as a Natural Therapeutic of Inflammatory Breast Cancer
    Dr. Michelle Martínez

The proposed study will investigate the in vivo anti-cancer effects of a dietary supplement containing whole mushroom compounds of Reishi on Inflammatory Breast Cancer (IBC) onset and progression. IBC is the most lethal and least understood form of advanced breast cancer. Its lethality originates from its nature of invading the vascular and lymphatic systems and absence of a typical tumor mass. Reishi compounds have been used in cancer to improve the immune system and kill malignant cancer cells. However, the effect of Reishi extract as a natural targeted therapeutic on IBC has not been investigated. Our preliminary data studying a human IBC cell line shows that whole mushroom Reishi extract inhibits IBC progression by affecting genes that contribute to cancer cell survival, invasion, metastasis, and progression. Reishi extract selectively inhibits IBC cell growth and invasion of the tumor environment. Furthermore, Reishi extract inhibits matrix metalloproteinase 2 and 9 secretion, and E-cadherin protein expression, possibly impairing the capacity of tumor spheroid formation that is characteristic of the IBC phenotype, and a requirement for invasion. Our HYPOTHESIS is that Reishi inhibits IBC onset and progression. Our long-term OBJECTIVES are to determine the therapeutic efficacy of whole mushroom Reishi extract, and investigate the molecular mechanisms by which Reishi affects IBC progression. SPECIFIC AIM 1 will determine the in vivo effects of whole mushroom Reishi extract on IBC onset and progression in an immunocompromised mouse model using novel imaging techniques. SPECIFIC AIM 2 will delineate the molecular targets of whole mushroom Reishi extract lymph nodes and primary tumors at the gene and protein level following treatment with placebo or Reishi extract. SPECIFIC AIM 3 will detail the mentoring and career developmental plans for the PI to achieve a successful independent research career. This proposal is UNIQUE in evaluating a role for a dietary supplement in a locally invasive breast cancer. This research is RELEVANT to public health because it promises to advance the understanding and detection of the therapeutic mechanisms of whole mushroom Reishi dietary supplement for this fatal disease and impact IBC patients, those at risk, and survivors of IBC.

  • The Role of Glial Monoamine Transporters in Cocaine-Induced Sensitization and Tolerance
    Dr. Mikhail Inyushin

In the United States, there are 1.5 million people classified as dependent on or abusing cocaine. Cocaine inhibits dopamine reuptake by binding to high-affinity monoamine transporters thereby impairing removal of dopamine from the synapse. These increased dopamine levels have been associated with the rewarding effect reported by cocaine abusers. In the brain, glial cells surround neuronal processes and synapses and have multiple types of monoamine transporters, including the organic cation transporters (OCT) that are not blocked by cocaine. The general hypothesis of the present proposal is that activation of astrocytes by repeated administration of cocaine enhances their OCT- mediated monoamine uptake thus decreasing dopamine levels in response to a drug challenge and leading to the development of tolerance to cocaine. This hypothesis will be tested in the following two specific aims:
Specific Aim 1: To determine the role of astrocytes in the development of behavioral sensitization and tolerance to cocaine. Our preliminary data suggest that inhibition of glial metabolism by the selective glial toxin fluorocitrate reduces tolerance to cocaine. We will determine if this effect is due to inhibition of monoamine uptake by the electrogenic transporters in astrocytes.
Specific Aim 2: To determine the effect of repeated cocaine and monoamine administration on transporter currents and the protein levels of OCT transporters in cultured astrocytes and rat brain. We will determine if elevated levels of monoamines, particularly dopamine, up-regulate expression of the OCT transporters on astrocytes resulting in apparent behavioral tolerance to cocaine. In the present electrophysiological and western blot experiments, we will assess the effects of monoamines on OCT electrogenic currents and protein levels in both cultured astrocytes (i.e., a direct effect) and in brain slices after rats have become sensitized or tolerant to cocaine.
Specific Aim 3: To determine the effects of repeated administration of cocaine on the accumulation in astrocytes of the fluorescent monoamine analog 4-(4-(dimethylamino)-styryl)-N-methylpyridinium; (ASP+). Using fluorescent imaging to study the accumulation of ASP+, a fluorescent substrate for DAT, NET and OCT in acutely separated astrocytes, we will measure the accumulation of ASP+ in acutely isolated astrocytes from control and cocaine treated animals.