Thomas J. Carew - US grants

During the last 16 years of this NSF-funded project, considerable progress has been made in using development as a powerful analytic tool for dissociating different forms of learning and memory in Aplysia on behavioral and cellular levels. These dissociations have in turn provided insights into both the similarities and differences in the mechanisms subserving different forms of learning and memory in this simple model system. In the present cycle period, we will continue to use this research strategy to investigate a fundamental question that has driven the project from the outset: the relationship between short-term and long-term memory. In addition, we will extend the project into a novel domain, examining the role of growth factors in development and learning in Aplysia.
Development of multiple memory phases in Aplysia
In this phase of the project we will examine the developmental assembly of several phases of memory: short-term, intermediate-term and long-term. This work will be carried out in both adult and developing animals on several interactive levels of analysis, including behavioral, synaptic and molecular levels. There are two primary objectives in this phase of the work: (i) to fully characterize the final adult phenotype in terms of memory processing capacity, and (ii) to analyze in detail the mechanistic assembly of memory processing during development.
The role of growth factors in development and learning
Many families of growth factors contribute importantly to developmental processes in vertebrate systems, and some of these factors are now clearly implicated in several forms of synaptic plasticity thought to contribute to learning and memory. We have recently found that specific growth factors can induce long-term synaptic changes in Aplysia sensory neurons and can regulate structural events in different types of Aplysia neurons in unique and cell-specific fashion. Thus in this phase if the project, using cellular, molecular and structural levels of analysis, we will examine the role of specific growth factors both in the developmental assembly of the central nervous system and in regulating memory formation in adult animals. A significant strength of this new direction is that the mechanistic roles of the growth factors in both development and learning can be directly analyzed and compared in the same experimental system.
The overall project will have a significant impact on advancing knowledge from two different perspectives. First, from a basic scientific perspective the project will elucidate fundamental cellular and molecular mechanisms used to induce lasting plasticity in both development and learning. Second, from an educational perspective insights derived from this project may be of basic utility in understanding (on a mechanistic level) the way in which our ability to learn and remember is influenced by critical developmental factors, and thus could be of considerable importance to the general field of education, considered in its broadest terms.

Development has proven to be a powerful analytic tool for dissociating different forms of learning and memory in Aplysia. In this project a developmental strategy will be used to explore two key questions: (1) What is the relationship between nonassociative and associative learning? Having already determined when nonassociative learning (sensitization) emerges, it will first be determined when associative learning (classical conditioning) emerges. Following this behavioral analysis, both forms of learning will be compared at several mechanistic levels: (i) biophysical; (ii) biochemical; and (iii) synaptic. (2) What is the relationship between short-term memory and long-term memory? Having already determined when short-term memory (minutes-hours) for sensitization emerges, it will first be determined when long- term memory (days-weeks) emerges. Following this behavioral analysis, the development of short-term and long-term memory will be investigated at biophysical, biochemical, and synaptic levels of analysis. By using development as a dissecting tool to uniquely dissociate and analyze complex behavioral and neuronal processes in a preparation such as Aplysia, which is highly suited for a mechanistic analysis, this research project can significantly advance scientific knowledge in an area of fundamental importance: the cellular and molecular mechanisms underlying learning and memory.***//

The marine mollusc Aplysia has been proven to be an extremely useful preparation for studying the cellular and molecular mechanisms of classical conditioning. We have recently discovered that Aplysia is also capable of exhibiting a reliabale form of operant conditioning: the animal rapidly learns to maintain a specific postural response in order to terminate the presentation of an aversive reinforcer (a bright light). Moreover, some of the elements in the neural circuit for the operant response are already known. Thus, it is the long term goal of this research project to analyze the cellular mechanisms of operant conditioning in Aplysia. There are two specific aims of the project: (1) a BEHAVIORAL ANALYSIS will be aimed at characterizing the detailed behavioral properties of the operant conditioning, including its basic and higher-order features; (2) a CELLULAR ANALYSIS will be aimed at specifying the neuronal mechanisms underlying the conditioning at progressively more fundamental levels, beginning with neural correlates in intact animals and progressing to directly producing the operant conditioning in both in vitro and analog systems, which permit detailed cellular investigation. Gaining insights into the cellular mechanisms of operant conditioning would be of significance from three perspectives: from a basic scientific perspective there is currently a real gap in our understanding of neuronal mechanisms of operant conditioning, even though it constitutes a significant form of associative learning; (2) from a theoretical perspective there has been a long-standing question in Psychology whether classical and operant conditioning represent two fundamentally different forms of learning. Since Aplysia now shows both forms of learning, a cellular analysis comparing their mechanisms could provide dey insights into this major theoretical question; and (3) from an applied perspective insights into cellular mechanisms of operant conditioning could potentially be of important clinical relevance. Since operant conditioning is routinely used in behavioral assessment of a wide variety of clinically relevant drugs, Aplysia could provide a powerful model system in which to explore the cellular actions of these drugs in mechanistic terms.

The long-term objective of this research project is to combine cellular and computational approaches in the analysis of information processing in an identified neural network which underlies a well analyzed behavioral response, the siphon withdrawal reflex, in the marine mollusc Aplysia. This simple reflex offers two key advantages for a computational analysis of learning: (1) the reflex exhibits a variety of forms of both non-associative and associative learning; and (2) the neural circuit underlying the reflex is quite well understood. Thus this system provides an excellent opportunity to construct a biologically realistic quantitative model, based on empirically determined cellular properties and synaptic interactions, that can provide important insights into the nature of information processing underlying learning and memory. There are three specific aims of the project: (1) a CELLULAR ANALYSIS will be directed at the detailed neuronal properties and synaptic interactions within the siphon withdrawal network; (2) a COMPUTATIONAL ANALYSIS will be directed at constructing a quantitative model that captures the essential features of information processing in the network; and (3) APPLICATIONS OF THE MODEL will be aimed at a quantitative analysis of specific forms of learning and memory known to be mediated by the network. Information obtained from this project would be of significance (1) from a basic scientific perspective since it could provide valuable insights into the cellular and molecular mechanisms underlying learning and memory; (2) from an applied perspective, since it could help to elucidate general computional principles utilized by a network to generate adaptive modifications (which in turn could facilitate construction of interactive machines capable of goal-directed behavior, for application in both clinical and educational contexts); and (3) from a theoretical perspective, since it could address fundamental questions concerning the nature of adaptive modifications within a computational network that are required for the expression of different forms of learning and memory.

The long-term objective of this research project is to identify the computational rules and design features that endow the central nervous system with its remarkable ability to acquire, store and retrieve information. To achieve this goal we will combine cellular and computational approaches in the analysis of information processing in a identified neural network which underlies a well analyzed behavioral response, the siphon withdrawal reflex (SWR), in the marine molluse Aplysia. This simple reflex offers two key advantages for a computational analysis of learning: (1) the reflex exhibits a variety of forms of both non-associative and associative learning; and (2) the neural circuit underlying the reflex is quite well understood. Thus this system provides an excellent opportunity to construct a biologically realistic quantitative model, based on empirically determined cellular properties and synaptic interactions, that can provide important insights into the nature of information process underlying learning and memory. There are three specific aims of the project: (1) a CELLULAR ANALYSIS will be aimed at examining the mechanisms of diverse forms of plasticity exhibited by individual, identified circuit elements: single cells and synapses; (2) a CIRCUIT ANALYSIS will be aimed at combining the elements studied at the first level into a functional network analysis, examining how the types of plasticity identified at individual circuit elements interact and contribute to both short-term and long-term plasticity exhibited within the SWR network; (3) APPLICATIONS OF THE MODEL will be aimed at examining how the different forms of cellular and network plasticity we have identified at the first two levels can account for actual behavioral instances of learning and memory in the SWR. Information obtained from this project would be of significance (1) from a basic scientific perspective, providing valuable insights into the cellular mechanisms underlying behavior and behavioral plasticity; (2) from an applied perspective, elucidating general computational principles utilized by a network to generate adaptive modifications such as signal optimizations and learning (which in turn could facilitate construction of interactive machines capable of goal-directed behavior, for application in both clinical and educational contexts) and (3) from a theoretical perspective providing information concerning the degree to which adaptive modifications required for learning within a neural circuit can be accounted for by cellular changes at specific loci on the one hand, or by distributed network processes on the other.

DESCRIPTION (provided by applicant): One of the great frontiers of modern neuroscience is the ongoing effort to understand how memory is processed in the brain. While the last decade has witnessed tremendous advances in our understanding of the synaptic and molecular basis of neuronal plasticity, much less is known about how the diverse forms of plasticity revealed in these studies directly relate to bone fide learning and memory. The main focus of this research project is to use the marine mollusc Aplysia to provide direct links between synaptic and molecular plasticity expressed on a cellular level, and specific phases of memory for sensitization expressed behaviorally. Towards that end, there are three specific aims to the overall project: (1) A BEHAVIORAL ANALYSIS will be aimed at elucidating the behavioral features of different phases of memory for sensitization (short-term, intermediate-term and long-term). The molecular induction and expression requirements of these forms of memory will also be examined in this phase of the project. (2) A SYNAPTIC ANALYSIS will be aimed at exploring, at a synaptic level, the different forms of memory revealed in behavioral studies, examining whether synaptic correlates of memory can be identified in the CNS, and whether behavioral memory can be blocked or enhanced by direct manipulations in the CNS. (3) A MOLECULAR ANALYSIS will be aimed at examining molecular correlates of different phases of memory, and establishing direct links between the induction of specific signaling cascades and activation of specific genes, and the induction of specific phases of memory. The importance of memory can perhaps be best appreciated by considering the devastating consequences of memory systems gone awry, such as the case in Alzheimer's Disease, Post-Traumatic Stress Disorder and victims of stoke or accidents that impair brain function. Thus understanding brain processes involved in memory formation is of fundamental importance both from a basic scientific perspective, with the goal of providing mechanistic insights into a fundamental aspect of cognitive behavior, and from a clinical perspective, with the goal of providing the basis for effective therapies for cognitive problems involving memory impairment.

[unreadable] DESCRIPTION (provided by applicant): This is a revised application for continued support of a multidisciplinary predoctoral program that focuses on training in the study of neural plasticity. We are requesting funds for ten trainees, exclusively at the predoctoral level. The program trains students to conduct high caliber research in neuroscience, specifically focusing on neural plasticity at the molecular, cellular, and systems levels. Neural plasticity is essential for basic neurobiological functions such as learning and memory, sensory processing, and synaptic modulation, and is critically impaired in many key mental and neurological disorders such as AIzheimer's disease and drug addiction. The participating training faculty include members of the Departments of Neurobiology and Behavior, Pharmacology, Anatomy and Neurobiology and Cognitive Science. All training faculty are broadly interested in neural plasticity. The faculty are recognized authorities in their fields, as evident by sustained productivity in research, scholarly awards, and research funding from federal agencies. The faculty are further distinguished by their extensive track record in providing predoctoral research training, as evidenced by 25 years of uninterrupted funding of this training grant (prior to this grant cycle). The program described in this proposal has been significantly modified, both in light of our own training goals and in response to the concerns of the reviewers. The modified program has the following features: During the first year, trainees complete a sequence of core courses ranging from molecular and cellular neurobiology to systems neuroscience to behavior. This core provides the trainee background for admission to the training program (in their second year). Students may choose to rotate through three different laboratories during the first year, or choose a home laboratory at the outset. In subsequent years, students complete four other advanced courses in subjects appropriate to the individual's interests, as well as supplementary courses in bioethics, statistics and writing. Full time thesis research follows the course work and laboratory rotations. Throughout their tenure, students are also involved in other training activities including teaching, journal clubs, seminars, and opportunities for data presentations. Trainees are evaluated for their ongoing progress in scholarship and research in quarterly meetings by the training faculty. An examination is required prior to advancement to candidacy as well as a dissertation defense for all predoctoral trainees. Our expected time to degree is 5-6 years. Our primary facilities are of two kinds. First, the facilities within individual laboratories specialized to the unique experimental questions examined therein. Second, core facilities which include (1) imaging (confocal and 2-photon), (2) a transgenic facility, (3) a genomic/microarray facility, (4) a proteomics facility (SELDI instrument), (5) a viral vector facility, and (6) a computer laboratory. Substantial effort has gone into making this program a highly interactive experience with extensive formal and informal contact between students, faculty, and visiting scholars. We believe that our revised training program provides an outstanding opportunity for predoctoral students interested in neural plasticity to gain critical experience in both basic scientific and clinical research. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Our ability to encode the events that occur in our world, store that information, and then retrieve it at a later time is essential for survival. Our memory serves the essential capacity to integrate past events into current adaptive behavior. Thus, understanding brain processes involved in memory formation is of critical importance from a basic scientific perspective, as it can provide mechanistic insights into a fundamental aspect of cognitive behavior. The overarching goal of this research project is to elucidate brain mechanisms of memory by using a powerful model system, the marine mollusk Aplysia, to forge direct links between neuronal plasticity expressed at the cellular and molecular levels, and specific phases of enduring memory for sensitization expressed behaviorally. To accomplish this goal, there are three Specific Aims: A BEHAVIORAL ANALYSIS will be aimed at identifying the "rules" that govern memory formation for a range of temporally discrete forms of memory for sensitization. Of special importance will be the unique roles of different patterns of training in the formation of mechanistically distinct forms of memory. A SYNAPTIC ANALYSIS will be aimed at establishing synaptic analogs of the different forms of memory revealed in the Behavioral Analysis, and determining the synaptic mechanisms that are recruited into play in forming these diverse memories. A MOLECULAR ANALYSIS will be aimed at identifying "molecular profiles" for different forms of memory by examining the molecular cascades (the genes, messages and proteins) that are involved in altering synaptic strength during memory formation. Relevance to public health: The importance of understanding brain mechanisms underlying memory can be especially appreciated in cases where memory is impaired, such as in Alzheimer's Disease, Post-Traumatic Stress Disorders, and in victims of accidents or strokes. Thus a major challenge in mental health is to achieve a basic understanding of the brain mechanisms that are engaged in normal memory formation, and how those mechanisms are impaired when memory is compromised by disease or injury. Such an understanding is essential for developing effective therapies for cognitive problems involving memory impairment. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The ability to learn, remember and recall information about the world is critical to every aspect of adaptive behavior. Despite striking recent advances in understanding neural mechanisms of memory, surprisingly little is known about how synaptic and nuclear events in neurons are coordinated in space and time in the induction of memory. This question forms the core of the present research program, which has the unique feature of combining the strengths of two independent laboratories, both of which utilize the marine mollusk Aplysia as a powerful preparation for studying the molecular basis of memory. The Carew group at UCI will contribute expertise in relating synaptic and molecular plasticity directly to bone fide learning and memory, and the Martin group at UCLA will contribute expertise in the analysis of signaling mechanisms between the synapse and the nucleus. The combined efforts of these two groups provide a unique opportunity to explore a fundamental question in cell biology, the mechanisms by which different compartments of a neuron communicate during memory formation. The project will be carried out at three interactive levels of analysis, captured in three Specific Aims: AIM I is a BEHAVIORAL ANALYSIS which will utilize a novel behavioral preparation that permits examining a monosynaptic sensory-motor (SN-MN) component of a reflex during intermediate-term and long-term memory formation, while simultaneously manipulating the local molecular environment of somatic and synaptic components of the reflex. AIM II is a SYNAPTIC ANALYSIS examining the SN-MN component of the reflex in the intact CNS in a preparation that allows specifying the contribution of somatic and synaptic events to synaptic facilitation. Here we will examine the mechanisms of (i) local induction of intermediateterm facilitation (ITF), (ii) synaptic induction of long-term facilitation (LTF), and (iii) conjoint synaptic and nuclear induction of LTF. AIM III is a MOLECULAR ANALYSIS examining the requirement for memory formation of (i) local translation at the synapse, focusing on two specific molecules, sensorin, a SN-specific neuropeptide, and cytoplasmic polyadenylation element binding protein (CPEB), a localized mRNA, (ii) facilitated importin-mediated transport of signals from synaptic compartments to the nucleus, and (iii) MARK signaling to the nucleus, CREB and C/EBP-mediated transcription, and induction of the immediate early gene C/EBP. Relevance to public health: Understanding the mechanisms whereby synapticallv generated signals trigger changes in gene expression in the nucleus during memory formation provides a means of identifying therapeutic targets for a variety of disorders including mental retardation, age-related memory loss and neuropsvchiatric diseases such as anxiety and mood disorders. [unreadable] [unreadable] [unreadable]

PROJECT SUMMARY/ABSTRACT A growing body of evidence suggests that growth factors (GFs), once considered to function mainly in development, also regulate synaptic plasticity and memory in the adult. This proposal reflects on an ongoing research program in our laboratory that has focused on the role of GFs in memory formation. The primary focus of this current project is to examine GF-mediated memory formation from a novel perspective, which takes into account the temporal aspect of their activity as part of the cellular computation involved in forming memories. Repeated-trial learning, a fundamental form of memory acquisition exhibited by virtually all animals, including man, requires a temporal interaction between an ongoing stimulus and the delayed effects of a previous stimulus. We will use a powerful paradigm to study repeated-trial training in the marine mollusk Aplysia, which develops long-term memory (LTM) for sensitization after only two training trials, but only if they are separated by a permissive and surprisingly specific time interval of ~45 min. This minimal system clearly separates the initiating stimulus (Trial 1) from the repeated stimulus (Trial 2), providing unparalleled access to the temporal interactions underlying repeated-trial LTM. We will investigate two GF-dependent mechanisms that our preliminary evidence suggests contribute significantly to temporal processing at the heart of repeated-trial learning. The first (explored in AIM 1) is a distributed mechanism for GF-dependent phosphorylation of extracellularly regulated kinase (ERK). The second mechanism (explored in AIM 2) is the multi-step signaling of a specific GF, TGF?, which we propose similarly integrates the timing of training trials. Finally, in AIM 3 we will try to establish causal connections between the timing of single ERK phosphorylation, TGF? availability, and the persistent effects of two-trial training The project holds promise for significant impact from both a basic scientific perspective and a clinical perspective. From a basic scientific perspective, Aplysia provides an exceptional experimental system that has the potential to demonstrate causal linkages between GF-mediated memory formation and its underlying synaptic and molecular mechanisms, while simultaneously exploring the temporal features of those mechanisms. And from a clinical perspective, the impact of this project addresses a major challenge in mental health: to understand and treat the devastating cognitive disorders that accompany neurodegenerative diseases. GF signaling has been directly implicated in many of these diseases and, since GFs are extracellular proteins, understanding when and where they act in the brain during memory formation could provide novel strategies for developing more specific and more effective therapeutic agents.