David L. Glanzman - US grants

The long-term objective of this project is to elucidate the cellular mechanisms of learning and memory. The withdrawal reflex of the marine snail Aplysia californica constitutes a useful model system for this purpose. First, the reflex exhibits several forms of learning, including short-term habituation, short- and long-term dishabituation/sensitization, and classical conditioning. Second, the neuronal circuit which mediates the withdrawal reflex is relatively well-understood, particularly the monosynaptic component of the circuit. Several short- and long-term plastic phenomena, which parallel, and may have mechanistic roles in behavioral modification of withdrawal, have been demonstrated at the sensorimotor synapse involved in withdrawal. Thus, the sensorimotor connection is a likely locus for learning-related cellular changes. Finally, the monosynaptic component of the withdrawal reflex can be reconstituted in dissociated cell culture. Such in vitro synapses greatly facilitate experimental dissection of the various cellular modifications, presynaptic and postsynaptic, which contribute to learning. These in vitro synapses are ideal, moreover, for studies involving optical recording and video microscopy. The proposed experiments utilize Aplysia sensorimotor synapses in dissociated cell culture for examining changes in morphology and in intracellular calcium, associated with plastic changes at this synapse. Calcium, because of its properties as a carrier of positive charge within neuronal membranes, as a trigger for exocytotic transmitter release, and as an activator of a variety of cytoplasmic and nuclear responses in neurons, is thought to play a significant role in synaptic plasticity. Optical recording techniques, together with video fluorescence microscopy, will be utilized to determine the specific changes in structure and in intracellular calcium that take place during a variety of forms of plasticity at in vitro sensorimotor synapses. In particular, changes in the dendritic structure of the motor neuron during long-term facilitation will be determined. How levels of calcium are altered in presynaptic varicosities or postsynaptic dendrites during homosynaptic depression, posttetanic potentiation, presynaptic facilitation, and activity-dependent facilitation will also be examined. Finally, the regulation of presynaptic transmitter release by the postsynaptic cell, as well as the cellular nature of this regulation and its implications for plasticity will also be studied. It is expected that the proposed research will significantly contribute to an understanding of the cellular substrates of learning and memory in nervous systems generally, and that the work may ultimately provide insights into memory-associated disease, such as Alzheimer's.

DESCRIPTION (provided by applicant): Perhaps the most basic form of associative learning, classical conditioning has been the subject of scientific investigation for a century. Nevertheless, the neurobiological mechanisms underlying classical conditioning remain poorly understood. The goal of the proposed research is to use a simple reflex that exhibits classical conditioning, and can be studied using reductionist neurobiological tools. Many of the neurons that underlie this reflex have been identified. In particular, the sensory and motor neurons for the reflex have been identified in the central nervous system (CNS). Moreover, the sensory and motor neurons can be individually dissociated from the CNS and placed into cell culture. This makes possible in vitro electrophysiological and molecular investigations of learning-related neuronal plasticity. In vitro studies of synaptic plasticity will be combined with studies of classical condititioning in semi-intact preparations that permit simultaneous electrophysiological and behavioral manipulation and measurement. A major focus of the proposed research will be on the roles of postsynaptic glutamate receptors, particularly NMDA and AMPA receptors, in classical conditioning. One potential mechanism for classical conditioning is modulation of the intracellular trafficking of AMPA receptors by the monoamine serotonin (5-HT). The cellular and molecular mechanisms of 5-HT-dependent modulation of AMPA receptor trafficking will be investigated in both neurons in culture and in the CNS. Pharmacological agents that block modulation of AMPA receptor trafficking will be used to identify the protein kinases involved in this modulation. Whether modulation of AMPAR receptor trafficking depends upon protein synthesis will also be studied. Immunohistochemical techniques will be used to label AMPA receptors, both in vitro and in the CNS. This will permit the direct visualization of changes in AMPA receptor distribution and number due to 5-HT and to learning. Furthermore, in situ hybridization will be used to determine whether long-term (>=24 hr) classical conditioning depends upon increased expression of genes for AMPA and NMDA receptors. Finally, the interactions between 5-HT-dependent processes and NMDA receptor-dependent processes during classical conditioning will be examined. The results of the project will clarify the basic neurobiology of learning, and will thereby facilitate the development of treatments for diseases of memory, such as Alzheimer's.

[unreadable] DESCRIPTION (provided by applicant): The purpose of this application is to provide the PI with resources that will enable him to acquire technical skills and expertise in molecular and cell biology. The PI is a Professor in the Departments of Physiological Science and Neurobiology at UCLA. During most of his scientific career the PI has studied the neural basis of simple forms of learning and memory in invertebrate organisms. The PI is highly trained in electrophysiological techniques for investigating learning-related neuronal plasticity, as well as in behavioral techniques for investigating simple forms of invertebrate learning and memory. Recent data from the laboratory of the PI indicates that the endogenous monoamine, serotonin (5-HT), modulates the intracellular trafficking of AMPA-type glutamate receptors in motor neurons of the marine snail Aplysia californica. Additional data from the Pl's laboratory suggests that 5-HT-induced modulation of AMPA-type receptor trafficking contributes to behavioral sensitization in Aplysia. To elucidate the potential cellular mechanisms of 5-HT-induced modulation of AMPA-type receptor trafficking in Aplysia, the PI wishes to acquire research skills in cellular and molecular biology. Specifically, during the award period the PI will undertake the following research projects: (i) to clone and sequence ionotropic glutamate receptor genes from the Aplysia nervous system; (ii) to perform in situ hybridization to localize the mRNAs of the cloned glutamate receptors in the Aplysia CNS; (iii) to express the Aplysia glutamate receptors in Xenopus oocytes for biophysical and pharmacological characterization of the expressed receptor channels; (iv) to generate antibodies to the different glutamate receptor subunits for use in immunohistochemical studies of the effect of 5-HT on the distribution of the receptors; and iv) to test whether expression of the glutamate receptor is enhanced in motor neurons of Aplysia during a form of long-term memory, long-term sensitization. To develop the technical expertise required for these projects, the PI will collaborate with several UCLA colleagues. The proposed collaborators are all highly knowledgeable and experienced in the molecular and cell biological techniques necessary for completing the proposed research. Furthermore, the laboratories of the collaborators have the necessary equipment for the proposed research.

[unreadable] DESCRIPTION (provided by applicant): An understanding of the neurobiological basis of learning and memory is one of the most important problems in modern neuroscience. At present there are several model organisms that are used to study learning and memory. No one organism, however, currently combines rapid embryological development, suitability for forwards and reverse genetic manipulation, electrophysiological tractability and a vertebrate genome and nervous system. The purpose of this proposal is to develop the zebrafish into a useful model organism for a cell biological analysis of learning and memory. The zebrafish has several advantages as a model organism for the study of learning and memory. It is suitable for both forwards and reverse genetics. In addition, the zebrafish possesses a simple reflexive escape response, the C-start, which is mediated by a relatively simple neural circuit in the fish's hindbrain and spinal cord. Many neurons within this circuit have already been identified and electrophysiologically recorded from. The C-start reflex will be used to investigate the genetic basis of simple forms of nonassociative and associative memory, including habituation, dishabituation, sensitization and classical conditioning. Both short-term and long-term learning protocols will be developed. Another major goal will be to develop reverse genetic tools for identifying genes that are important for learning and memory in the zebrafish. Toward this end a methodology for disrupting the expression of specific genes (gene knockdown) using RNA interference (RNAi) will be developed. In addition, a computational screen will be developed to identify learning-associated genes in the zebrafish. Such genes will then become targets for gene knockdown in studies of learning and memory. It is expected that the findings from this study will speed the development of the zebrafish into a model organism for memory studies. In addition, the results from the proposed experiments will contribute to a fundamental understanding of the processes that underlie learning and memory. Such an understanding will facilitate the development of treatments for Alzheimer's disease and other disorders of memory. [unreadable] [unreadable] [unreadable]

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).

A major unsolved problem in science is how the brain stores and recalls information. While significant progress has been made in recent decades, much work remains to be done in order to achieve a complete understanding the neural basis of learning and memory. A significant impediment toward such an understanding is the vast complexity of the mammalian brain. For this reason it would be useful to develop a simpler model system in which to study fundamental biological mechanisms of learning and memory. During the project behavioral and electrophysiological investigations of simple forms of learning in the zebrafish (Danio rerio) will be carried out. The zebrafish, although a vertebrate organism, exhibits several reflexive behaviors that are mediated by relatively simple neuronal circuits in its hindbrain and spinal cord. The project will focus on the startle reflex in the larval zebrafish. Attempts will be made to identify specific changes in the central nervous system of the zebrafish, particularly changes in the strength of synaptic connections between neurons that mediate three basic forms of learning: habituation, sensitization and classical conditioning. The underlying motivation of these investigations is that an understanding of the neural basis of learning in the larval zebrafish will provide important insights into learning and memory in more complex organisms, including humans. The project will provide unique training opportunities for a postdoctoral investigator, as well as for undergraduate students working in the laboratory.

DESCRIPTION (provided by applicant): A significant percentage of people in the US have disorders of long-term memory; among these are people suffering from Alzheimer's disease (AD), posttraumatic stress disorder (PTSD), diabetes and depression. In addition to problems forming new memories, patients suffering from these diseases can have difficulty accessing older memories, especially in the advanced stages of the diseases, or-in the case of PTSD-patients may have difficulty regulating traumatic long-term memories. An important, and at present unresolved, question is the extent to which the memory difficulties experienced by some patients stem from retrieval problems or to degradation of the physical memory traces themselves. In order to answer this question, neuroscientists must learn more about how the brain maintains long-term memories. Contrary to long-held beliefs, older memories are not stable, even in healthy individuals, but, under some circumstances, can be rendered strikingly labile. Furthermore, once in this labile state the memories can become permanently disrupted. Two phenomena of long-term memory, termed memory reconsolidation and memory erasure, have attracted particular attention in this regard. Memory reconsolidation refers to the finding that, after having been given a reminder cue for a previously learned experience, a previously consolidated memory of that experience can become disrupted by treatments, such as exposure to inhibitors of protein synthesis, that interfere with original memory consolidation. So- called memory erasure has been observed following inhibition of a specific isoform of protein kinase C (PKC), known as PKM¿. PKM¿ contains the catalytic domain of an atypical PKC, but lacks the regulatory domain and is therefore constitutively active. This property, it has been proposed, endows PKM¿ with the capacity mediate memory maintenance. The phenomena of memory reconsolidation and memory erasure remain poorly understood and controversial. In part, these problems stem from the enormous complexity of the mammalian brain, as well as the complexity of the forms of memory that have been examined in studies of reconsolidation and memory erasure. This project will develop a simple model experimental system for a reductionist analysis of memory reconsolidation and memory erasure. The focus of the project will be on a nonassociative form of learning, long-term sensitization (LTS), in the marine snail, Aplysia. This organism offers several major advantages for the study of long-term memory maintenance, including the ability to investigate a form of long-term (>24 hr) synaptic plasticity, known as long-term facilitation, that unambiguously mediates the learning. The PI will use behavioral, cellular and molecular techniques to determine the neurobiological mechanisms that underlie memory reconsolidation and memory erasure. Data from the proposed studies will facilitate the development of treatments for disorders of long-term memory, including AD and PTSD.