Jack W. Lipton, Ph.D. - US grants

DESCRIPTION (adapted from applicant's abstract): In previous research, the investigators have shown that fetal rat brain cocaine levels can vary by as much as 400 percent as a function of uterine position following a single injection to the dam. They hypothesized that fetuses exposed to low levels of cocaine as a result of their position in the uterus would be less likely to exhibit teratogenesis, resulting in greater variability in the experimental group, and increasing the likelihood of Type I (false negative) errors. This may explain why there is such an abundance of equivoval research reports with respect to animal studies. They subsequently demonstrated that prenatal exposure to cocaine produced reductions in CNS (striatal) and peripheral (carotid body) dopamine (DA) as well as the important DA developmental growth factor glial cell line-derived neurotrophic factor (GDNF) in a positionally-dependent fashion. Suprisingly, those fetuses with the lowest brain cocaine levels exhibited the most severe reductions in DA and GDNF. This led them to presume that cocaine was producing its effects through vasoconstriction which they confirmed by demonstrating that cocaine accentuates the cervical to ovarian gradient of vasoconstriction normally extant within the uterus. Based on all of these findings they have hypothesized that cocaine induced uteroplacental vasoconstriction (CIUV) leads to fetal oxidative stress, attenuating the production of GDNF which reduces the maturation and function of the mesencephalic DA system, the magnitude of which depends upon the fetus's relative uterine position. The current research was designed to determine if CIUV results in measurable oxidative stress in the fetus and whether these measures are correlated with striatal and carotid body alterations in GDNF and DA in a positionally-dependent fashion. The successful completion of these studies will allow them to clearly understand the role uterine position plays in the interpretation of animal data and whether CIUV produces damage to neuronal tissues that result in defined reductions in GDNF and DA. Only after such studies are completed can investigators make an informed judgement regarding the potential liability of prenatal cocaine exposure in animals and its importance in understanding the complex and subtle cognitive and behavioral deficits only now being observed in the rapidly maturing, prenatally cocaine-exposed, human populations.

[unreadable] DESCRIPTION (provided by applicant): Recently, consumption of MDMA or 'ecstasy' has been rising among young women. Consequently, there is a substantial risk of exposure among women who are, or become, pregnant while abusing MDMA. The few prenatal MDMA animal studies conducted have failed to demonstrate any lasting neurobiological alterations from prenatal MDMA exposure. Alternative models utilizing neonatal rather than prenatal MDMA exposures have demonstrated alterations in serotonergic systems as well as learning decrements, but the validity of neonatal rodent models is questionable as they model 3rd trimester development (pregnant women use MDMA primarily in the 1st trimester). We have recently demonstrated that prenatal MDMA exposure from days 14-20 induced long lasting changes in DA metabolism at postnatal days (P) 3, 7 and 21 as well as increased DA fiber density when examined at P21. At P21 DA fiber density in prenatally MDMA exposed rats was increased in the frontal cortex by 502% with smaller but significant increases in striatum, nucleus accumbens and the lateral septum. This was the first published report that prenatal MDMA can result in long-lasting brain alterations. We propose to longitudinally examine the persistence of these neurochemical and histological changes in prenatally MDMA exposed rats as well as examine changes in DAT and trophic factors that may provide a mechanism for the observed changes in DA neurochemistry and TH fiber density. Stereologic cell counts of TH+ neurons in the SN and VTA revealed no changes in cell number, suggesting that the increased fiber density is a result of collateral sprouting rather than increased cell numbers. We have also demonstrated increased fiber degeneration at P2 (silver stain) followed by signals for neurite collateral sprouting at P7 (netrin-l/ephrin-b2). Bolte-Taylor et al (1998) [5] demonstrated that lesions of the Raphe result in 5-HT fiber loss in the cortex followed by a proliferation of DA fiber collaterals. Based upon these findings we hypothesize that MDMA induced losses of 5-HT fibers as a result of prenatal MDMA exposure is the mechanism behind increased TH fiber density in the frontal cortex as well as other structures. Furthermore, we will examine whether our prenatally MDMA-exposed, DA hyperinnervated rats are more sensitive to the neurotoxic potential of methamphetamine, a known DA neurotoxin. Ultimately, these studies should provide us with specific answers with respect to the importance of 5-HT fiber degeneration, monoamine fiber competition, trophic factors and sprouting cues on our observed changes in the brain of MDMA exposed neonates and whether such subjects are at greater risk for damage from environmental neurotoxic insults as they mature. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Among adolescents and college students, 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy") use has steadily grown, while the use of many other stimulants has declined or stabilized. Recently, consumption has been rising among young women [74]. Consequently, there is a substantial risk of exposure among women who are, or become, pregnant while abusing MDMA. Recently, we have demonstrated that prenatal MDMA exposure from embryonic days 14-20 (E14-20) induced long lasting decreases in DA and 5-HT turnover and increases in tyrosine hydroxylase (TH) fiber density [42]. TH fiber density in prenatally MDMA exposed rats was significantly and dramatically increased in the prefrontal cortex (PFC: 502%) as well as in striatum (STR: 9%), nucleus accumbens (NAc: 8%) and the lateral septum (LS: 15%) at P21. Stereologic cell counts of TH+ neurons in the substantia nigra (SN) and ventral tegmental area (VTA) revealed no changes in cell number, suggesting that the increased fiber density is a result of axonal sprouting rather than increased cell numbers. We therefore hypothesized that target derived pro-growth cues, specifically axonal guidance molecules or trophic factors, are changed in subjects prenatally exposed to MDMA and that such cues are the mechanisms whereby MDMA achieves this significant morphological increase in TH fiber density. In support of this hypothesis we have demonstrated that prenatal MDMA results in significant mRNA and protein increases in the axonal guidance cues netrin-1 and ephrin-b2 within the striatum and nucleus accumbens. These molecules govern axon innervation patterns via regulation and orientation of axonal growth. Collectively, these findings are the first to demonstrate that prenatal MDMA exposure results in persistent alterations in the development of the neonatal brain. The aims of this application are designed to determine the extent and time course of the development of axonal guidance cues, trophic factors and consequent increases in TH fiber density in DA-rich target regions (specifically the striatum and prefrontal cortex) after prenatal MDMA exposure. In addition, by utilization of the brain slice culture system, we can examine the consequences of the direct application of MDMA on the expression of these guidance and growth cues in an in vitro source-target system. [unreadable] [unreadable] These studies will provide a sound basis for future experiments into the consequences of prenatal MDMA on the developing and aging brain. In addition, they will provide a basis for pharmacologic interventions aimed at minimizing the profound alterations in axonal sprouting, guidance and connectivity that we have already begun to observe experimentally. [unreadable] [unreadable]

Core B: Goals Al. In order to provide consistency and the highest quality of catecholamine quantitation, Core B will provide all sub-projects with well-controlled analytical chemistry services via high performance liquid chromatography with electrochemical detection (HPLC-EC) employing state-of-the-art coulometric array detection. A2. Core B will provide relative gene-expression quantitation via Real Time RT-PCR using both low density arrays, as well as quanititation of individual genes as directed by sub-project investigators. In addition, Core B will also be a resource to troubleshoot develop and run ELISAs and/or western blots to determine whether key changes in gene expression result in concomitant changes in protein expression as needed. A3. Core B will provide both single- and double-labeling in situ hybridization and analyses of both film and emulsion autoradiography. This will afford determination of both regional localization and semi-quantitative analysis of mRNA expression as well as cellular resolution for determination of cellular phenotype. A4. Finally, surgical services from all of the projects have been moved into Core B. This will allow Core B to provide surgical services for the 6-OHDA lesion studies associated with all of the subprojects. Funneling subjects of all projects through Core B to provide consistent surgical services will further enhance the Center's ability to provide cross project quality control. RELEVANCE (See instructions): The purpose of this core application is to provide support services for a large center focused on examining problems and potential therapeutic solutions for Parkinson's disease. The core will conduct animal surgeries, quantify molecules from tissues of those animals, analyze the results of such experiments and communicate those results back to the project leaders.