The Role of the VTA NMDA Receptors, VTA DA Cells and VTA Terminal Regions in Reward-Related Learning

Reward-related learning occurs when an initially neutral stimulus acquires the capacity to elicit responses similar to an unconditioned stimulus (US) with which it is associated, in which case the stimulus now functions as a conditioned stimulus (CS). The mechanisms whereby stimuli come to function as CSs are not fully understood and comprise the theme of this dissertation. We have previously proposed that coincident signals from an unconditioned and the eventual conditioned stimulus (US and CS) signals on dopamine (DA) cells of the ventral tegmental area (VTA) leads to strengthening of CS synapses, allowing the CS to acquire the ability to activate VTA DA cells on its own and elicit conditioned approach, thereby functioning as a CS. Furthermore, we proposed that this type of learning is VTA NMDA receptor dependent. This dissertation was designed to test this model by specifically testing the following hypotheses: (1) A food US will activate VTA DA cells; (2) A food-associated CS will activate VTA DA cells and cause conditioned approach; (3) Blockade of NMDA receptors in the VTA will prevent a food-associated stimulus from acquiring the capacity to function as a CS (i.e., cause conditioned approach) and to cause conditioned activation of the mesocorticolimbic DA system. To test the hypothesis that a US, in this case food, activates VTA DA cells, male rats were maintained at 85% of their free feeding weights for the duration of this study. Rats were exposed to an eating protocol in which the rats were able to eat or not eat food. Rat brains were then removed and immunostained for c-Fos followed by tyrosine hydroxylase (TH) to examine VTA DA cell activation. As expected, rats that ate the food demonstrated a significantly greater number of VTA DA (TH-labeled) cells expressing c-Fos than rats that did not receive food. To test the hypothesis that a CS, in this case a food-associated light, activates VTA DA cells, male rats were maintained at 85% of their free feeding weights for the duration of this study. Rats were trained to retrieve a food pellet after a light presentation (the CS) and then tested for the expression of the food checking response with only CS presentations. As expected, a light functioning as a CS caused a significantly greater number of VTA DA cells to express c-Fos than a light not functioning as a CS. We also hypothesized that VTA NMDA receptor stimulation is necessary for a food-associated stimulus (CS; also a food-associated light) to elicit conditioned approach via conditioned activation of VTA DA cell terminal regions (mesocorticolimbic DA terminal regions). Rats were prepared with indwelling cannulae positioned so as to allow bilateral microinjections of AP-5 (a NMDA receptor antagonist) in the VTA and were maintained at 85% of their free feeding weights. Male rats were exposed to an acquisition or expression of learning protocol. Subsequently, all rat brains were processed for c-Fos labeling to examine activation of mesocorticolimbic DA terminal regions. As expected, AP-5 significantly impaired the acquisition of conditioned approach and significantly reduced the amount of c-Fos expressed in the cells in the mesocorticolimbic DA terminal regions in response to the CS. Also, AP-5 did not impair the expression of the already learned conditioned approach response. All together, the results support the model - that a CS acquires, via the VTA NMDA receptor, the capacity to cause conditioned activation of VTA DA cells and mesocorticolimbic DA terminal regions therefore eliciting conditioned approach in a manner similar to a US

Determining Effective Methadone Doses for Individual Opioid-Dependent Patients

BACKGROUND: Randomized clinical trials of methadone maintenance have found that on average high daily doses are more effective for reducing heroin use, and clinical practice guidelines recommend 60 mg/d as a minimum dosage. Nevertheless, many clinicians report that some patients can be stably maintained on lower methadone dosages to optimal effect, and clinic dosing practices vary substantially. Studies of individual responses to methadone treatment may be more easily translated into clinical practice. METHODS AND FINDINGS: A volunteer sample of 222 opioid-dependent US veterans initiating methadone treatment was prospectively observed over the year after treatment entry. In the 168 who achieved at least 1 mo of heroin abstinence, methadone dosages on which patients maintained heroin-free urine samples ranged from 1.5 mg to 191.2 mg (median = 69 mg). Among patients who achieved heroin abstinence, higher methadone dosages were predicted by having a diagnosis of posttraumatic stress disorder or depression, having a greater number of previous opioid detoxifications, living in a region with lower average heroin purity, attending a clinic where counselors discourage dosage reductions, and staying in treatment longer. These factors predicted 42% of the variance in dosage associated with heroin abstinence. CONCLUSIONS: Effective and ineffective methadone dosages overlap substantially. Dosing guidelines should focus more heavily on appropriate processes of dosage determination rather than solely specifying recommended dosages. To optimize therapy, methadone dosages must be titrated until heroin abstinence is achieved

Differential Expressions of the Alternatively Spliced Variant mRNAs of the µ Opioid Receptor Gene, OPRM1, in Brain Regions of Four Inbred Mouse Strains

The µ opioid receptor gene, OPRM1, undergoes extensive alternative pre-mRNA splicing in rodents and humans, with dozens of alternatively spliced variants of the OPRM1 gene. The present studies establish a SYBR green quantitative PCR (qPCR) assay to more accurately quantify mouse OPRM1 splice variant mRNAs. Using these qPCR assays, we examined the expression of OPRM1 splice variant mRNAs in selected brain regions of four inbred mouse strains displaying differences in µ opioid-induced tolerance and physical dependence: C56BL/6J, 129P3/J, SJL/J and SWR/J. The complete mRNA expression profiles of the OPRM1 splice variants reveal marked differences of the variant mRNA expression among the brain regions in each mouse strain, suggesting region-specific alternative splicing of the OPRM1 gene. The expression of many variants was also strain-specific, implying a genetic influence on OPRM1 alternative splicing. The expression levels of a number of the variant mRNAs in certain brain regions appear to correlate with strain sensitivities to morphine analgesia, tolerance and physical dependence in four mouse strains

High-throughput behavioral phenotyping in the expanded panel of BXD recombinant inbred strains

Genetic reference populations, particularly the BXD recombinant inbred (BXD RI) strains derived from C57BL/6J and DBA/2J mice, are a valuable resource for the discovery of the bio-molecular substrates and genetic drivers responsible for trait variation and covariation. This approach can be profitably applied in the analysis of susceptibility and mechanisms of drug and alcohol use disorders for which many predisposing behaviors may predict the occurrence and manifestation of increased preference for these substances. Many of these traits are modeled by common mouse behavioral assays, facilitating the detection of patterns and sources of genetic coregulation of predisposing phenotypes and substance consumption. Members of the Tennessee Mouse Genome Consortium (TMGC) have obtained phenotype data from over 250 measures related to multiple behavioral assays across several batteries: response to, and withdrawal from cocaine, 3,4-methylenedioxymethamphetamine; “ecstasy” (MDMA), morphine and alcohol; novelty seeking; behavioral despair and related neurological phenomena; pain sensitivity; stress sensitivity; anxiety; hyperactivity and sleep/wake cycles. All traits have been measured in both sexes in approximately 70 strains of the recently expanded panel of BXD RI strains. Sex differences and heritability estimates were obtained for each trait, and a comparison of early (N = 32) and recent (N = 37) BXD RI lines was performed. Primary data are publicly available for heritability, sex difference and genetic analyses using the MouseTrack database, and are also available in GeneNetwork.org for quantitative trait locus (QTL) detection and genetic analysis of gene expression. Together with the results of related studies, these data form a public resource for integrative systems genetic analysis of neurobehavioral traits

Mu and Delta opioid receptors activate the same G proteins in human neuroblastoma SH-SY5Y cells

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72251/1/sj.bjp.0704430.pd

The AIDS and Cancer Specimen Resource: Role in HIV/AIDS scientific discovery

The AIDS Cancer and Specimen Resource (ACSR) supports scientific discovery in the area of HIV/AIDS-associated malignancies. The ACSR was established as a cooperative agreement between the NCI (Office of the Director, Division of Cancer Treatment and Diagnosis) and regional consortia, University of California, San Francisco (West Coast), George Washington University (East Coast) and Ohio State University (Mid-Region) to collect, preserve and disperse HIV-related tissues and biologic fluids and controls along with clinical data to qualified investigators. The available biological samples with clinical data and the application process are described on the ACSR web site. The ACSR tissue bank has more than 100,000 human HIV positive specimens that represent different processing (43), specimen (15), and anatomical site (50) types. The ACSR provides special biospecimen collections and prepares speciality items, e.g., tissue microarrays (TMA), DNA libraries. Requests have been greatest for Kaposi's sarcoma (32%) and non-Hodgkin's lymphoma (26%). Dispersed requests include 83% tissue (frozen and paraffin embedded), 18% plasma/serum and 9% other. ACSR also provides tissue microarrays of, e.g., Kaposi's sarcoma and non-Hodgkin's lymphoma, for biomarker assays and has developed collaborations with other groups that provide access to additional AIDS-related malignancy specimens. ACSR members and associates have completed 63 podium and poster presentations. Investigators have submitted 125 letters of intent requests. Discoveries using ACSR have been reported in 61 scientific publications in notable journals with an average impact factor of 7. The ACSR promotes the scientific exploration of the relationship between HIV/AIDS and malignancy by participation at national and international scientific meetings, contact with investigators who have productive research in this area and identifying, collecting, preserving, enhancing, and dispersing HIV/AIDS-related malignancy specimens to funded, approved researchers at no fee. Scientific discovery has been advanced by this unique biorepository. Investigators are encouraged to browse the ACSR Internet site for materials to enhance their own scientific initiatives

The Effects of Tail Biopsy for Genotyping on Behavioral Responses to Nociceptive Stimuli

Removal of a small segment of tail at weaning is a common method used to obtain tissue for the isolation of genomic DNA to identify genetically modified mice. When genetically manipulated mice are used for pain research, this practice could result in confounding changes to the animals' responses to noxious stimuli. In this study, we sought to systematically investigate whether tail biopsy representative of that used in standard genotyping methods affects behavioral responses to a battery of tests of nociception. Wild-type littermate C57BL/6J and 129S6 female and male mice received either tail biopsies or control procedural handling at Day 21 after birth and were then tested at 6–9 weeks for mechanical and thermal sensitivity. C57BL/6J mice were also tested in the formalin model of inflammatory pain. In all tests performed (von Frey, Hargreaves, modified Randall Selitto, and formalin), C57BL/6J tail-biopsied animals' behavioral responses were not significantly different from control animals. In 129S6 animals, tail biopsy did not have a significant effect on behavioral responses in either sex to the von Frey and the modified Randall-Selitto tests of mechanical sensitivity. Interestingly, however, both sexes exhibited small but significant differences between tail biopsied and control responses to a radiant heat stimulus. These results indicate that tail biopsy for genotyping purposes has no effect on nocifensive behavioral responses of C57BL/6J mice, and in 129S6 mice, causes only a minor alteration in response to a radiant heat stimulus while other nocifensive behavioral responses are unchanged. The small effect seen is modality- and strain-specific

Human matrix metalloproteinases: An ubiquitarian class of enzymes involved in several pathological processes

Human matrix metalloproteinases (MMPs) belong to the M10 family of the MA clan of endopeptidases. They are ubiquitarian enzymes, structurally characterized by an active site where a Zn(2+) atom, coordinated by three histidines, plays the catalytic role, assisted by a glutamic acid as a general base. Various MMPs display different domain composition, which is very important for macromolecular substrates recognition. Substrate specificity is very different among MMPs, being often associated to their cellular compartmentalization and/or cellular type where they are expressed. An extensive review of the different MMPs structural and functional features is integrated with their pathological role in several types of diseases, spanning from cancer to cardiovascular diseases and to neurodegeneration. It emerges a very complex and crucial role played by these enzymes in many physiological and pathological processes