Ischemic brain injury is a major cause of death and disability with few effective treatments. Spreading depression (SD) can enhance brain injury if it occurs less than a day before ischemia. However, SD also can reduce injury if it occurs 1-3 days before ischemia, so-called ischemic tolerance. Thus, neural cells and tissues have an endogenous ability to modulate the extent of their own injury. SD itself is noninjurious. Furthermore it induces astrogliosis over the same time period as SD- induced ischemic tolerance. Since glia vitalize neurons, SD-induced gliosis may be a key transformation needed for the development of SD- induced tolerance to ischemic injury. Accordingly, the general goal of this proposal is to define the fundamental signaling mechanisms responsible for SD and so begin to define the basic triggers needed for SD-induced gliosis and SD-induced tolerance to excitotoxic injury. Experiments will systematically combine an innovative in vitro preparation with modern investigative tools to explore fundamental physiologic and anatomic changes of SID. Hippocampal organ cultures (HOTCs) will be used throughout this project because: (A) the HOTC is an intact area of brain tissue that maintains many cell-to-cell relationships found in vivo yet HOTCs survive in vitro for months; (B) microenvironmental conditions of HOTCs can be easily controlled; (C) individual cells within the HOTC can be followed in space and time; (D) we have shown that HOTCs support SD and develop astrogliosis like that seen in vivo. Thus, SD, SD-induced gliosis and SD-induced tolerance which require days to evolve, can now be examined for the first time in vitro. This means that the experimental advantages of easy access and microenvironmental control of in vitro preparations can be applied to answer 'mechanistic' questions at the cellular and molecular level of events that require intact tissues. Patch clamp technology, computer- based imaging strategies, and molecular biologic techniques will be used to accomplish the following specific aims. (1) Examine the spatiotemporal dynamics of gap junction functional changes between neurons and between astrocytes associated with SD. (2) Examine the spatiotemporal changes and identity of SD-induced currents in hippocampal pyramidal cells. (3) Examine the mechanisms of cellular and interstitial Ca2+ changes of SD. (4) Examine how modulation of Ca2+ or acid-base changes of SD influence SD-induced gliosis and tolerance to excitotoxic injury.