John P. Walsh - US grants

The influence of age on long-term synaptic plasticity will be studied int he striatum using in vitro brain slice and intracellular recording techniques. Synaptic efficacy will be monitored by sampling responses to paired stimuli. This procedure allows simultaneous monitoring of short- term, presynaptic changes in release and long-term tetanus induced changes in synaptic efficacy. Correlations will then be performed between paired- pulse and long -term synaptic plasticity. Each hypothesis will be tested under conditions that increase the probability of inducing LTD and, in a separate set of experiments, LTP. Initially the effect of senescence will be established for LTD and LTP. The second specific aim will then test the hypotheses that 1) inrinsic dopamine (DA) released in in vitro slices modulates synaptic plasticity in the striatum, and 2) that this modulation declines with age. DA will be depleted through unilateral 6-OHDA lesions to the substantia nigra (SNc). Lesion effects will be determined through comparisons to the contralateral (DA intact) striatum. DAergic modulation of synaptic plasticity will then be studied DA perfusion of slices ipsilateral to the 6-OHDA lesion. Since intrinsic release will be eliminated in these slices, dose-response comparisons can be made between age-groups. This procedure thus allows us to test the hypothesis that age-dependent differences in DA receptor activation alter DA modulation of synaptic plasticity. DA depletion will be quantified by calculating loss of tyrosine hydroxylase positive neurons in the SNc and measurements of DA content int he striatum (HPLC). The third specific aim will test the hypotheses that 1) long-term synaptic plasticity is triggered by tetanus induced transients in intracellular Ca2+ and that 2) this process is altered by age-related changes in Ca2+ homeostasis. Two complementary procedures will be used to differentiate between the role of pre- and postsynaptic fluxes of Ca2+. First, brain slices will be exposed to a low Ca2+ solution that will reduce Ca2+ influx uniformly in pre-and postsynaptic compartments. These result will be compared with 1) data from slices bathed in normal Ca2+ and 2) data obtained when only postsynaptic Ca2+ is lowered through intracellular injection of BAPTA. The fourth specific aim will determine if synaptic plasticity int he striatum is related to cell classification and location in the calbindin D28K defined 3-dimensional patch-matrix system. The final goal of this proposal will be to determine if age-related alterations in motor performance are correlated with underlying deficits in striatal synaptic plasticity. In total, these studies will provide a novel electrophysiological framework for interpreting basal ganglia-related changes in motor performance in aging.

The experiments outlined in this project will test hypotheses that 1) extend findings on age-related changes in striatal physiology and 2) examine the localization and modulation of nigral Ca2+ currents. These studies will blend morphological and physiological disciplines and apply them to in vitro brain slice, acute neuronal isolation and embryonic mesencephalic culture techniques. Sharp electrode and whole-cell patch clamp techniques will be used for physiological analysis. The theme of the first set of studies is an investigation of mechanisms underlying changes in short-term synaptic plasticity and synaptic reorganization in the aged striatum. Synaptic efficacy and failure rate will be determined through 1) responses to minimal stimulation and 2) an analysis of 4-AP induced spontaneous EPSPs. Postsynaptic striatal cells will be filled with biocytin and examined ultrastructurally to look for relationships between synaptic structure and synaptic plasticity. Lastly, synapses will be experimentally eliminated and the influence of age on reinnervation will be examined physiologically. The theme of the second set of studies is to examine the relationship between dendrites and somatically measured Ca2+ signals. Cortical and nigral lesions will be used to test the hypothesis that synapse elimination induces a reactive dendritic regression that is expressed physiologically as a decrease in the duration of striatal plateau potentials. A second set of experiments will examine the relationship between dendrites and Ca2+ signals by characterizing somatically recorded Ca2+ currents during the postnatal period of dendritic expansion. Acute isolation methods will then be used to examine the impact of dendritic pruning of somatic Ca2+ signals. The third set of studies will test the hypotheses that neurotropins act on dopaminergic substantia nigra neurons through a modulation of 1) Ca2+ currents and 2) afferent synaptic efficacy.

DESCRIPTION (provided by applicant): The goal of the proposed research is to determine the underlying mechanisms for short-term and long lasting changes in striatal synaptic function created by transient mitochondrial inhibition. My laboratory has demonstrated that aged rats consistently show a loss of facilitating synapses and, remarkably, that low doses of 3-nitropropionic acid (3-NP) (single i.p. injection, 20 mg/kg) caused an enduring change in corticostriatal synaptic function that mimics this synaptic phenotype of aging. To gain insight into the mechanisms underlying this enduring 3-NP-mediated change in synaptic function, we examined rats 24 hours after the injection and found an opposite trend: a dramatic increase in the expression of long-term potentiation (LTP). Experiments outlined in Specific aim 1 will examine the time course of 3-NP induced changes in corticostriatal synaptic function to provide data about the transition from 3-NP induced LTP to long-term depression (LTD), as well as new information about how long the 3-NP induced increase in corticostriatal synaptic depression lasts. Experiments outlined in Specific Aim 2 will investigate the role played by NMDA receptor and dopamine (DA) receptor activation in the 3-NP-mediated enhancement of LTP expression 24 hours after 3-NP exposure. Experiments outlined in Specific Aim 3 will examine the role played by reactive oxygen species (ROS) in mediating 3-NP induced changes in synaptic function. While it is clear ROS are involved in changes associated with mitochondrial dysfunction, the striatum is unique in the potential interaction between ROS and (DA) physiology. Experiments outlined in Specific Aim 4 will examine the role played by DA and its oxidation in the expression of 3-NP induced changes in synaptic function. Parallel analyses of changes in DA biochemistry and in the numbers of DA receptors will be performed to gain greater insight into how changes in DA physiology contribute to 3-NP induced changes in corticostriatal synaptic function. The experiments outlined in Specific Aim 5 will determine if the 3-NP-induced changes in corticostriatal plasticity can generalize to reversible inhibition of complex II by malonate. In total, information gained from these experiments will provide new insight into mechanisms of synaptic change resulting from mitochondrial inhibition as well as a correlative framework for similar mechanisms, which may occur in aging and disease.

Dopamine-glutamate plasticity in nigrostriatal injury: Exercise-enhanced recovery Principal Investigators: Michael Jakowec, PhD, and John Walsh, PhD. University of Southern California. The primary goal of this research proposal is to elucidate the molecular mechanisms underlying the interactions between dopaminergic and glutamatergic neurotransmission and the role that intensive exercise plays in mediating recovery following injury to the nigrostriatal dopaminergic neurons by the neurotoxicant MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine). Previously, in our studies in the MPTP-lesioned mouse model of dopamine depletion we discovered that high-intensity treadmill running, started 5 days post-MPTPlesioning when cell death is complete and continued for 28 days, altered corticostriatal synaptic plasticity in dopaminergic and glutamatergic neurotransmission within the basal ganglia and that these alterations resulted in significant improvement in the recovery of motor performance. We found that motor improvement is not simply due to changes in the total level of striatal dopamine as measured by HPLC analysis, but rather due in part to alterations in dopamine release from remaining nigrostriatal terminals, which is accompanied by increased expression of the dopamine D2 receptor. In addition, both molecular and electrophysiological studies of striatal medium spiny neurons indicated altered glutamatergic neurotransmission specifically increased expression of GluR2 subunits, an important member of the AMPA subtype of glutamate receptor. This proposal will utilize a novel transgenic mouse strain termed BAC-D2-eGFP in which green fluorescent protein is expressed exclusively within the indirect dopamine D2 receptor containing projection neurons of the basal ganglia to determine if exercise-induced changes are pathway specific. Mice will either be administered MPTP or saline and a subset from each group subjected to intensive treadmill exercise for 28 days. This proposal consists of two specific aims.

This project is developing an interactive online multimedia teaching tool (OMTT) to be used by an interdisciplinary community of professors and students in undergraduate neurosciences, gerontology, psychology, bioengineering and biology courses, and with the intention of improving the understanding of complex science processes by a wide range of students. The objectives of the project are to:

1. enhance STEM learning for a group of diverse undergraduate science students by making course material easier to understand and more accessible,
2. demonstrate to students the connection that exists between basic science research and science education, and
3. improve recruitment and retention of students in the sciences by building a diverse scientific community and encouraging student interaction with faculty.

The OMTT is expected to enhance learning by providing students with continuous online access to course material used in class by their professors. The interactive multimedia presentation of course content includes scrolling text, video, narration and computer animations, thus offering students self-paced access to varied and rich representations of the curriculum. Additionally, the OMTT educates students about ongoing research and recent scientific publications as they relate to class topics. For each topic, students can explore a written description of a professor's research laboratory, a short video illustrating how research is performed to address the issue taught in class, and examples of articles published by the professor. This feature is meant to improve student understanding of the connection between core subject material and the research that creates this knowledge base, and to facilitate student contact with professors in the field and identify research opportunities. The OMTT also includes on-line assessments that can be used by students and professors to gauge student learning, and are intended to enable early intervention for struggling students.

Students have the opportunity to become digital scientists by completing digital laboratory exercises imbedded in learning exercises in the online multimedia-teaching tool. The OMTT is designed to be easily modified and adapted by faculty in other disciplines and at other institutions. Enhancement of the infrastructure and broad dissemination is occurring through multiple avenues, including those based at the University of Southern California and via the Internet.

The Use of Multimedia, Social Media and Gaming to Teach Neuroscience Via Mobile Devices project creates an innovative online STEM educational platform for delivering undergraduate neuroscience curriculum that optimizes the use of multimedia, game psychology and social networking. The expected outcome of this novel approach to neuroscience education is an increase in student engagement, an expansion of the instructive reach of the professor beyond the classroom, and an improvement in student retention of course material. The web platform is all-inclusive and equipped to handle an entire semester's curriculum and promotes a classroom experience that enhances student engagement, affinity and knowledge in neuroscience beyond that achieved by traditional instructional methods. The use of HTML5 web design is key to use and dissemination of this project, since it ensures access from any computer or mobile device, regardless of its operating system. This extends the learning experience outside the classroom and it optimizes the use of open access educational multimedia that is available online by enhancing coordinated delivery. Professors are able to customize the integration of open access materials within the framework of their own course design. HTML design also guards against changes in web standards through its backwards compatibility. Social networking technologies used in the platform improve the learning process by fostering student collaboration and co-learning through familiar communication technologies. The game-mechanics in the platform immerse students in the course through reward and engagement mechanisms built into the game design. Ultimately, the neuroscience instructional platform creates a site for faculty and student development which illustrates best-practices for both instructors and students on how to effectively use it, including a blog for discussing success and failure.

Impact on teachers and students is tested directly in a large general education (GE) course using proper statistical methods to evaluate the online platform's merit to science instruction. The principal investigator operates a neurophysiology research laboratory at USC, and is the professor in charge of implementing the proposed neuroscience education platform into his large GE course in neuroscience. Worldwide access by students, as well as professors who are seeking ways to modernize their own science courses, is enhanced by the immediate accessibility online offered by the platform employed in this project. Moreover, the project platform advances the potential for networking with developers of online science education tools, thus increasing collaboration and utilization of online educational resources developed through support by government grants and private foundations. The neuroscience course developed and the platform it is constructed into serves as a test case for easy-to-use platforms designed for instructors to create their own mobile-based learning environment for their field of STEM education.