Microinjection of Rat Brain Synaptoemma into Xenopus oocytes (2012 - Present)

Investigator:  Steven Symington, Salve Regina University
Mentor:  J. Marshall Clark, University of Massachusetts - Amherst 

Abstract:  Current approaches to toxicity testing are time consuming, expensive and typically rely on investigations that evaluate observable changes to whole animals. New approaches capable of assessing environmental contaminants in a cost and time efficient manner are required to provide information necessary for sound evaluation of the health effects of adverse environmental agents. Newly developed approaches must also be able to assess chemical mixtures, different life stages and species, while reducing the use of animals. The research proposal outlined below utilizes well-established neurotoxicants to evaluate the potential of microtransplantation of native mammalian tissue with endogenous ion channels into Xenopus oocytes. Xenopus oocytes, injected with the synaptolemma from rat brain, will allow the direct and comparative action of pyrethroids to be determined in vitro on multiple target sites in a functional system. Incorporation of intact lipid rafts, with native ion channels and auxiliary proteins supporting neurotransmitter release, provides a physiologically-relevant system that couples the electrophysiological means to study channel gating with the functional and biochemical complexity of intact presynaptic nerve terminals. Individual ion channel targets will be isolated using electrophysiology protocols, buffer composition changes, and pharmaceutical agents. Synaptolemma, isolated from specific brain regions, including pre- and post-natal rats, will allow sensitivity comparisons to be made spatially and temporally. If successful, this protocol is amenable to the study of developmental and reproductive toxicity of pyrethroids and to the study of any agent causing acute, chronic and developmental neurotoxicity in mammalian neuronal tissues, including those from knockout mice and humans. The specific aims of the present proposal are; 1) Isolate and characterize ion currents, membrane potential and neurotransmitter release from synaptolemma-injected oocytes; 2) Validate the in vivo relevance of oocytes injected with synaptolemma from pyrethroid sensitive and insensitive brain regions; 3) Determine if a direct age-dependent neurotoxicity exists for pyrethroids using oocytes injected with synaptolemma from different developmental stages.