Emmanuel M. Landau - US grants

Alcoholism is a major public health problem in the U.S.. Research into its interaction with the brain is far from conclusive. We propose to investigate the effects of ethanol on the locus coeruleus and on one of its major target areas, the hippocampus, because this system has been implicated in other types of addiction (opiates) and may have a major role in determining mood and modulating anxiety. Experiments will be conducted on tissue slices in vitro. Ethanol and other drugs will be introduced into the superfusing medium in known quantities. Iontophoretic and intracellular injection of drugs will also be done when indicated. Neurons will be impaled with glass micro-electrodes, and the membrane voltage amplified and measured, using standard electrophysiological techniques. Membrane resistance will be measured with a bridge arrangement and the cells will at times be voltage clamped using a one-electrode voltage clamp device. The slice will be stimulated directly to elicit synaptic potentials. We will be examining three major research questions: l) The effects of ethanol on the membrane properties and synaptic input characteristics of the neurons. These will include the membrane potential, membrane resistance, spike amplitude and threshold, the response to stimulation of synaptic inputs and the effects of bath applied transmitter substances; 2) The effects of ethanol on the calcium dependent potassium current, which is a major determinant of neuronal excitability. We will attempt to elucidate how ethanol interacts with intracellular calcium and cyclic nucleotides to affect this current; 3) The effects of ethanol will be compared with a number of membrane fluidizing agents to test the hypothesis that membrane fluidization is the molecular mechanism of action of ethanol.

Alcoholism is a major public health problem in the U.S., but research into interaction with the brain is far from conclusive. We propose to investigate the effects of ethanol in the hippocampus and some of its inputs, in particular, the locus coeruleus. These structures are involved in regulating mood, anxiety and memory and are involved in other types of addiction (opiates). Experiments will be conducted on tissue slices in vitro. Ethanol and other drugs will be introduced into the superfusing medium in known concentrations, or applied iontophoretically. Cells will be impaled with glass micro-electrodes for intra-cellular recording and stimulation, and also will be voltage clamped using a one-electrode voltage clamp amplifier. Synaptic potentials will be elicited by extra- cellular stimulation. The following major research questions will be posed; 1) What is the mechanism of the dual effect of ethanol on neuronal excitability? Our work to date indicates that ethanol may both increase and decrease excitability. We propose to test a hypothesis according to which both effects are due to increased phophatidyl inositol degradation induced by ethanol, the suppression of excitability being due to the attendant changes in inositol tri-phosphate and the increases in excitability being caused by activation of protein kinase C. 2) Does ethanol affect any other protein kinase C sensitive mechanisms? We propose to study the effects of ethanol on a number of protein kinase C related mechanisms in hippocampus, locus coeruleus and dorsal raphe. This research program proposes to build on our existing excitability data and to follow a recent biochemical lead, namely the increased hydrolysis by ethanol of phosphatidyl inositol, to search for novel effects of ethanol. In the process we hope to gain insight into the role the phosphatidyl inositol system plays in the intoxication and addiction to ethanol.

DESCRIPTION: (Applicant's Abstract) Long-term potentiation (LTP) is a cellular memory process which is the focus of intense interest. Most attention has so far been devoted to the role of synaptic kinases in this process. In view of the burgeoning interest in the cellular role of phosphatases, the place of these enzymes in the mechanism of LTP is of great relevance. As has been shown in a recent publication from our group, postsynaptic phosphatases act as a gate for LTP, which is controlled by the cAMP pathway. The postsynaptic phosphatases are arranged in a cascade involving calcineurin, protein phosphatase 1 (PP1) and inhibitory protein 1 (I-1). I-1 is the point where the cascade is inhibited by cAMP. In the present application it is proposed to further define the phosphatase cascade involved in LTP, by applying specific inhibitors of calcineurin and PP1, as well as biochemically measuring the phosphorylation of I-1 after high frequency synaptic stimulation (HFS). It is also proposed to investigate the mechanism for the influence of stimulation pattern on the cAMP-dependence of LTP which may be mediated by postsynaptic accumulation of calmodulin. Finally, the role of phosphatases and cAMP in the maintenance phase of LTP will be studied. The proposed experiments are based on a detailed and testable model, and on solid preliminary data. The importance of LTP for understanding memory processes, the fundamental importance of placing postsynaptic phosphatases in the theoretical framework for LTP, and the ubiquity and importance of cAMP signaling in the brain, all lend significance to the proposed project.

DESCRIPTION (provided by applicant): Neuronal plasticity is thought to underlie a variety of behavioral processes, including memory formation and addiction. Long-term potentiation (LTP) is a persistent, use-dependent form of neuronal plasticity exhibited by synapses in the hippocampus, a brain structure that has been implicated in memory formation and retrieval. Hippocampal LTP has emerged as the major model system for studying the cellular basis of behavioral memory. Long-lasting forms of LTP require protein synthesis (translation). However, little is known about the relationship between LTP and the regulation of translation. The experiments of this proposal address this issue by determining the role in LTP of several important translation control points, which are regulated through protein phosphorylation. We will use synaptic stimulation to induce persistent LTP in rat hippocampal slices. These slices will then be analyzed with biochemical and immunohistochemical methods to determine the distribution and phosphorylation of proteins that participate in translation initiation. Experiments will test for the activation of ribosomal S6 kinase and the phosphorylation of S6, a protein of the 40S ribosomal subunit that selectively promotes the translation of mRNAs containing a terminal oligopyrimidine (TOP) tract in their 5' untranslated regions. Other studies will investigate the possible involvement of the translation initiation factor elF4E and its regulatory binding partner, 4E-BP, which preferentially regulate the translation of transcripts containing structured 5' ends. The cellular mechanisms underlying the regulation of these initiation factors will be studied by inhibiting enzymes of the translation initiation pathway as well as more upstream signaling pathways. Finally, a set of immunohistochemical experiments will study the distribution of translation-related phosphoproteins in the dendritic region using quantitative confocal microscopy. The results of this proposal will shed light on the mechanisms that participate in drug addiction, as well as learning and memory, by providing new information on the role of translation control in neuronal plasticity.