Brain worsens or lessens the severity of injury to itself by means that include altered synaptic activity (SA) that likely involves inflammatory mediators (IM's) since they have both pro- and anti-inflammatory effects on injury plus equally opposite effects on SA. Such divergent, net effects likely involve differential patterns and stimulus dependency of the production, expression and subsequent effects of IM's in brain tissue which is regionally and cellular heterogeneous. However, confirming this notion has previously been impossible due to the absence of appropriate tools that would allow needed simultaneous measurements of multiple candidate molecules from identified cells and the interstitial space (ISS) within functioning brain tissue. We propose to remove this void by formally coupling 2 new investigative tools for measurement of IM protein changes in identified cells (and ISS) from rodent hippocampal organ cultures (HOTC's) and age-matched whole animal controls. Multiple and simultaneous measurements of targeted IM's will be made using the Bio-Plex Protein Array System and specific cell-type samples derived from a Leica laser dissection microscope. Our general goal is to establish detailed protocols for simultaneous measurement of multiple proteins from identified cells plus the ISS and then illustrate the biologic utility for their use with the exemplary SA change seen with seizures. This project will be focused via 3 important steps to more clearly illustrate its potential impact. First, we will alter SA through the use of seizures, since this stereotypic perturbation of brain induces changes in IM's. Second while many IM's change with seizures, we will examine interleukin-1 (IL-1), a prototypic IV. Third, 6 key IL-1 family IM's will be examined since understanding their simultaneous behavior is essential to eventually deciphering how IL-1 effects SA and brain function. IL-1 family IM's consist of 3 receptor ligands (IL-1alpha, IL-1beta & IL-lreceptor antagonist (Ra)), 2 receptor subtypes (IL-1RI & IL-1RII) and an accessory protein (IL-1AcP). While elevated levels of IL-1alpha and IL-1beta enhance seizures, the soluble form of IL-1 Ra (slL-1Ra) acts as an anticonvulsant.
Our specific aims are: 1: Develop and confirm protocols for simultaneously measuring IL-1 family IM in identified cells and the ISS from HOTC's and hippocampus in vivo. 2: Determine the spatiotemporal and cell-specific pattern of IL-1 IM changes induced by seizures in hippocampus in vivo and in HOTC's. 3: Determine how excitability conditioning (i.e., from late-long-term potentiation (L-LTP) and exogenous alteration of IL-1 IM homeostasis) alters IL-1 IM expression, seizure susceptibility & injury in HOTC's and whether these changes are interdependent. Accurately determining the where, when and to what degree IL-1 family IM's simultaneously change in identified cells is consistent with the R21 program and should speed deciphering how IL-1 family IM's cause dual effects on seizures and SA.
| Kunkler, Phillip E; Hulse, Raymond E; Schmitt, Michael W et al. (2005) Optical current source density analysis in hippocampal organotypic culture shows that spreading depression occurs with uniquely reversing currents. J Neurosci 25:3952-61 |
| Hulse, R E; Kunkler, P E; Fedynyshyn, J P et al. (2004) Optimization of multiplexed bead-based cytokine immunoassays for rat serum and brain tissue. J Neurosci Methods 136:87-98 |
