Arnold Eskin - US grants

A remarkable property of cells is their ability to generate endogenous rhythms with extremely long periods in the 24 hr range. To express this property, cells contain a circadian oscillating mechanism coupled to an output system. Also, environmental information must be coupled to some element of the circadian oscillating mechanism to account for the normal regulation or entrainment of circadian rhythms. Our long-term goals are to understand the mechanisms of entrainment and generation of circadian oscillators. The objectives of the proposed research are to identify proteins that play a role in the circadian system and to discover the regulatory mechanisms that link the proteins together to form an oscillatory network. The isolated eye of Aplysia contains a circadian pacemaker that is regulated over two distinct entrainment pathways. Previously, we have used light and serotonin (5-HT) as probes to define elements of the entrainment pathways and to identify protein candidates for components of the circadian oscillator. The first aim of our research is to determine the function of proteins that are putative components of entrainment pathways or the oscillating mechanism. We will explore the function of important proteins by. obtaining amino acid sequences of peptides obtained from protein spots of preparative 2D-gels. Only after the functions of these proteins are known can precise models of oscillator components be proposed and tested. Recently, we identified one putative oscillator protein of Aplysia as a lipocortin-like protein. The second aim of our research is to investigate the role of this lipocortin-like protein in the circadian oscillator. First, we will test the hypothesis that the lipocortin-like protein regulates arachidonic acid metabolism and thus affects other oscillating elements through second messengers produced from arachidonic acid. Secondly, we will use antibodies of the lipocortin-like protein to localize the protein and study its variability. The third and fourth aims are to investigate the role of transcription and protein kinase activity in the regulation and generation of the circadian oscillator. The results of our experiments will identify important proteins in the circadian system and suggest relationships between the proteins. Also, the functions of several. putative protein components of the circadian system will be revealed. Finally, the importance of transcription, kinase activity, and arachidonic acid metabolism in circadian timing will be elucidated.

A remarkable property of cells is their ability to generate endogenous rhythms with extremely long periods in the 24 hr range. To express this property, cells contain a circadian oscillating mechanism coupled to an output system. Also, environmental information must be coupled to some element of the circadian oscillating mechanism to account for the normal regulation or entrainment of circadian rhythms. Our long-term goals are to understand the mechanisms of entrainment and generation of circadian oscillators. The objectives of the proposed research are to identify proteins that play a role in the circadian system and to discover the regulatory mechanisms that link the proteins together to form an oscillatory network. The isolated eye of Aplysia contains a circadian pacemaker that is regulated over two distinct entrainment pathways. Previously, we have used light and serotonin (5-HT) as probes to define elements of the entrainment pathways and to identify protein candidates for components of the circadian oscillator. The first aim of our research is to determine the function of proteins that are putative components of entrainment pathways or the oscillating mechanism. We will explore the function of important proteins by. obtaining amino acid sequences of peptides obtained from protein spots of preparative 2D-gels. Only after the functions of these proteins are known can precise models of oscillator components be proposed and tested. Recently, we identified one putative oscillator protein of Aplysia as a lipocortin-like protein. The second aim of our research is to investigate the role of this lipocortin-like protein in the circadian oscillator. First, we will test the hypothesis that the lipocortin-like protein regulates arachidonic acid metabolism and thus affects other oscillating elements through second messengers produced from arachidonic acid. Secondly, we will use antibodies of the lipocortin-like protein to localize the protein and study its variability. The third and fourth aims are to investigate the role of transcription and protein kinase activity in the regulation and generation of the circadian oscillator. The results of our experiments will identify important proteins in the circadian system and suggest relationships between the proteins. Also, the functions of several. putative protein components of the circadian system will be revealed. Finally, the importance of transcription, kinase activity, and arachidonic acid metabolism in circadian timing will be elucidated.

The major objective of this research is to gain insights into the biochemical mechanisms involved in the induction, mediation and maintenance of long-term sensitization as well as the interrelationship between the short- and long-term forms of this simple example of nonassociative learning. To address these issues we will utilize the tail-siphon withdrawal reflex in Aplysia. Key components of this reflex and its plasticity are the large homogeneous clusters of bilaterally symmetrical sensory neurons in the pleural ganglia. Our specific focus will be on the identif- ication and characterization of proteins and their regulation in sensory neurons, an understanding of which is a prerequisite to elucidate cellular mechanisms of learning and memory in this model system. The proposal has four specific aims. First, identify proteins whose synthesis and/or phosphorylation are altered by sensitization training and by treatments that mimic the effects of sensitization training. Although we will be screening for changes of all proteins, we will pay particular attention to proteins involved in the metabolism of second messengers. We will try to select, for further study, proteins that are behaviorally relevant. Second, determine the temporal relationship between protein changes and the induction treatments. Third, identify mechanisms that produce changes in the proteins. Fourth, identify functions of proteins altered by induction treatments. research will extensively use quantitative analysis of proteins separated by two dimensional gel electrophoresis and peptide sequencing. This research will provide substantial new insights into the mechanisms for the induction, storage and read-out of a form of long-term memory as well as help address fundamental questions regarding the memory. Moreover, the research will provide general information on the role of specific proteins and second messenger systems in cellular function and plasticity.

The long-term goal of this research is to understand how changes in properties of neurons lead to the formation of memory and subsequent changes in behavior. The behavioral framework of our research is long-term sensitization of the defensive tail, siphon withdrawal reflex of Aplysia. Glutamatergic sensory neurons are key components of the reflex. Long-term presynaptic facilitation (LTF) at the sensory-motor synapse is mainly responsible for sensitization. We hypothesize that a number of properties of a sensory neuron must change in a coordinated fashion to produce LTF and store memory. Thus, we predict that the increase in transmitter release that occurs during LTF should be coordinated with an increase in uptake and synthesis of glutamate. In testing this hypothesis, we found that sensitization training leads to a substantial long-term increase in the Vmax of the high affinity neuronal glutamate transporter. In addition, we found that glutamine uptake was increased suggesting that glutamate synthesis was also increased. These findings provide the basis for molecular and biochemical studies on newly-discovered plastic properties of the sensory neuron. In particular, we will study molecular correlates of memory storage and expression in Aplysia and will extend these findings to research on long-term potentiation in the hippocampus of rats. The proposed research has 4 Specific Aims: Aim 1 is to determine the relationship between increases in glutamate uptake, glutamate synthesis, and behavioral sensitization. Aim 2 is to determine the cellular mechanisms that mediate coordinate regulation of glutamate uptake, synthesis, and long-term facilitation. Aim 3 is to determine whether the mechanisms responsible for the long-term increase in glutamate uptake involve synthesis of transporters and then transport through the ER-Golgi-vesicle transport pathway. Aim 4 is to determine the generality of regulation of uptake by studying glutamate uptake during long-term potentiation in the CA1 region of the hippocampus. Deficiencies in glutamate uptake have been implicated in a number of diseases (e.g., ALS, Hodgkin's, Alzheimer's). Glutamate appears to be a remarkably potent and rapidly-acting neurotoxin. This underlines the important role of glutamate uptake in the normal functioning of brains. Our research will help elucidate possible causes for decreased glutamate uptake in these diseases by identifying mechanisms responsible for long-term regulation of glutamate transporters.

The aim of this proposal is to acquire state-of-the-art instrumentation that will form the foundation for a Molecular Analysis Center at the University of Houston. The requested BIAcoreTM 2000 will advance studies addressing DNA replication, learning and memory, circadian rhythms, antibody/antigen recognition, enzyme structure and mechanism, enzyme design, control of RNA stability, assembly and maintenance of nerve myelin, DNA structure, and biotechnology, by facilitating the identification and/or quantification of the diverse biomolecular interactions occurring during these processes.

A number of properties of a neuron change in a coordinated fashion to store a given type of memory. Our long- term goal is to determine which properties of neurons change, and how these properties are regulated during formation of memories. Regulation of glutamate transporter activity may be necessary during increases in synaptic efficacy to maintain the fidelity of synaptic transmission and to avoid toxicity that might occur if glutamate is elevated in the synaptic cleft for too long. Thus, we hypothesize that increases in glutamate transporter activity will accompany increases in synaptic efficacy at glutamatergic synapses, especially ones involving long-term changes in synaptic efficacy. This hypothesis will be tested in vitro by investigating regulation of glutamate uptake after induction of LTP and in vivo by investigating regulation of glutamate uptake after contextual fear conditioning. Area CA1 of the rat hippocampus will be used in most experiments. We will use a multidisciplinary approach including electrophysiology, biochemistry and behavioral analysis to investigate the regulation of glutamate uptake. The proposed research has four specific aims. Most of the experiments in Specific Aims 1-3 will investigate regulation of glutamate transport during two different forms of LTP (E- and L-LTP). Aim 1 is to characterize the mechanisms involved in the increase in glutamate uptake produced by high frequency stimulation. Aim 2 is to determine whether induction of LTP increases the expression of glutamate transporters, and what mechanisms are involved in the increase in expression. Aim 3 is to investigate the relationship between LTP and changes in glutamate uptake and expression of glutamate transporters. Aim 4 is to determine whether an associative learning paradigm, contextual fear conditioning, produces an increase in glutamate transport in the hippocampus in vivo. In our lab, recent studies have shown that glutamate uptake is regulated during long-term memory in Aplysia, and preliminary results indicate that glutamate uptake is regulated during LTP in the rat hippocampus as well. Thus, the results of these studies will likely indicate that regulation of glutamate transport is a general phenomenon at glutamatergic synapses involved in synaptic plasticity. As glutamate is a remarkably potent and rapidly acting neurotoxin, fundamental studies of long-term regulation of glutamate transport should aid solutions to brain trauma and diseases such as amyotrophic lateral sclerosis.

DESCRIPTION (provided by applicant): Understanding how learning and memory are modulated is an important aspect of understanding how memories are formed. Emotional state, various environmental factors, and diseases of the nervous system can impact the formation of memory. The long-term goals of our research are to define factors that normally modulate memory formation and to determine how these factors modulate learning and memory. In addressing these goals, we discovered that the circadian clock modulates a simple form of long-term memory, long-term sensitization of the tail-siphon withdrawal reflex in Aplysia. Animals form long-term memory when they are trained during the day but form much less memory when they are trained at night. However, short-term memory of the same behavior is formed equally well during the day and night. The Specific Aims extend our previous research by investigating circadian modulation of other forms of memory and the mechanisms responsible for circadian modulation of long-term memory. Specific Aim 1 is to determine the scope and function of circadian modulation of learning and memory. We will investigate circadian modulation of a more complex associative form of learning (learning that food is inedible), and we will investigate circadian modulation of learning in nocturnal species of Aplysia. Specific Aim 2 is to determine the circuitry involved in the circadian modulation of learning and memory. We will investigate the role of the circadian pacemaker in the eye, and we will determine whether circadian modulation of memory persists in a semi-intact preparation. Specific Aim 3 is to determine whether the circadian clock modulates learning and memory through modulation of facilitatory serotonergic neurons in the learning and memory circuitry. We will investigate whether circadian modulation of release of serotonin is responsible for the modulation of memory formation. Specific Aim 4 is to determine which biochemical and molecular events necessary for the induction of long-term memory in sensory neurons are responsible for the circadian modulation of memory. We will investigate circadian modulation of synthesis of ApC/EBP mRNA and activation of MAPK and PKA during the formation of memory. In addition to elucidating the modulation of learning and memory, this research will provide insights into how the circadian clock regulates its outputs to produce rhythms. Furthermore, knowing the mechanisms by which memory formation is modulated will aid attempts to improve memory as well as to treat diseases that affect memory.

DESCRIPTION (provided by applicant): Plasticity of glutamatergic synapses is important in the storage of information in the brain. Neuronal and glial plasma membrane glutamate transporters play major roles in removing released glutamate from the synaptic cleft. Although the function of glutamate transporters has received considerable attention, only recently has it been demonstrated that glutamate uptake is regulated during expression of long-term synaptic plasticity. Glutamate uptake is increased during long-term sensitization in Aplysia, during LTP in the hippocampus of the rat, and during morphine addiction and withdrawal in the rat. These findings suggest that long-term regulation of glutamate uptake during changes in synaptic efficacy may be a phylogenetically conserved mechanism that occurs along with different types of synaptic plasticities. Furthermore, deficiencies in glutamate uptake have been implicated in a number of diseases including ALS, epilepsy, and Alzheimer's. Our long-term goal is to elucidate the general principles and functions of regulation of synaptic glutamate uptake associated with synaptic plasticity. Our specific aims address the induction, expression, maintenance, and function of long-term changes in glutamate uptake that occur as a result of sensitization training in Aplysia. The proposed research has 4 specific aims: Aim 1 is to characterize the activity of Aplysia glutamate transporters expressed in sensory neurons and Xenopus oocytes. In this aim, we will study the properties of an Aplysia glutamate transporter (ApGT1) expressed in oocytes following injection of ApGT1 cDNA. Aim 2 is to determine the induction mechanisms that produce the long-term increase in glutamate uptake. We will study the roles of TGF-beta and C/EBP in mediating the long-term effects of sensitization training. Aim 3 is to determine the mechanisms of expression/maintenance that are responsible for the increase in glutamate uptake. We will study whether sensitization training alters levels of ApGT1 mRNA, levels of ApGT1 protein, and synthesis of ApGT1 protein. We will also investigate the half-life of ApGT1 protein. Aim 4 is to determine the physiological role of regulation of glutamate uptake. We will study modulation of regulation of glutamate uptake and test the hypothesis that the function of the increase in glutamate uptake is to reduce postsynaptic receptor desensitization. In addition to elucidating mechanisms of induction, expression, and maintenance, our results will elucidate physiological roles of regulation of glutamate uptake and set the stage for research aimed at discovering the molecular mechanisms that couple induction processes to expression processes during learning and memory formation