In this proposal we describe immunohistochemical methods for visualizing, at the light and electron microscopic level, neural pathways in which lithium alters phosphoinositide metabolism by inhibiting myo-inositol-l-phosphatase. Those methods will also be developed into quantitative procedures for measuring the tissue levels of inositol phosphates for which, in some cases, there exist no methods and where in other cases, the methods are extremely time consuming, limiting research on the substances. The research has importance at two related levels. The first is that lithium inhibition of myo-inositol-l-phosphatase may be the source of one aspect of lithium's efficacy in the treatment of mania. Thus in the presence of pharmacologically meaningful levels of lithium, stimulated neural cells in the CNS undergo rapid and large decreases in myo-inositol, an effect that may reduce the responsivity of receptors dependent on phosphoinositides for their function. The other important aspect of this study is that one of the inositol phosphates that we will develop methods to measure is known to be released from phosphatidylinositol-4,5-bisphosphate in response to receptor stimulation and to act as a second messenger, the role of which is to mobilize intracellular Ca2+. That substance is myo-inositol-1,4,5-trisphosphate a substance for which no method for quantitative measurement exists, thus hampering research, especially in vivo studies, on its tissue levels and their changes under controlled conditions. Thus our proposed methods will identify neural pathways that are sensitive to lithium's effect and, in this context, pathways that use calcium mobilization as part of the chain of events that leads to their response. Described in the application are methods for preparing immunogens to most of the known inositol phosphates produced following receptor activation. Those immunogens will be used to raise antibodies specific for each of the inositol phosphates and those antibodies will then be used to develop immunoassays for those substances and immunohistochemical tissue staining procedures to visualize their presence morphologically.
