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.

9410579 Glanzman The goal of this project is to explain the mechanisms of learning and memory in an invertebrate animal, the sea slug Aplysia. The sensory and motor neurons which mediate certain simple defensive withdrawal reflexes of Aplysia can be removed from the animal's nervous system and grown in cell culture; here they will reform their original connections (called synapses). These synapses in cell culture are ideal for analyses of the mechanisms of learning- related changes in the strength of the synapses. Recently, these researchers found that the synapses formed between Aplysia sensory and motor neurons in culture exhibit a long- term change similar to that which occurs at synapses in the vertebrate nervous system and which is thought to mediate associative learning. This synaptic change is known as "long-term potentiation" (LTP). This project will analyze the cellular mechanisms of LTP in the Aplysia synapses in cell culture and determine whether LTP of sensorimotor synapses mediates classical conditioning of Aplysia's withdrawal reflex in the whole animal. The hope is that an understanding of how the relatively simple nervous system of Aplysia mediates learning will help advance our understanding of how the far more complex nervous systems of mammals mediate more sophisticated forms of learning and memory.

LAY ABSTRACT IBN-9808930 Changes in the strength of connections (synapses) between neurons in the brain are believed by most neuroscientists to underlie learning and memory. Synapses in the brain may increase in strength, or decrease in strength, as a consequence of learning by an animal. One prominent form of a change in synaptic strength that occurs in the mammalian brain is known as homosynaptic long-term depression or LTD. LTD is a decrease in the strength of a synapse, lasting 60 minutes to hours, produced by low-frequency stimulation of a presynaptic neural pathway. Many synapses in a part of the brain known as the hippocampus exhibit LTD. Because the hippocampus plays an important role in learning and memory in mammals, it is thought that LTD might represent a neural mechanism of memory. Despite a half-decade of work on LTD in the hippocampus, however, relatively little is understood about the cellular mechanisms that underlie LTD. Furthermore, even less is understood regarding what role LTD of hippocampal synapses might play in learning and memory. Part of the difficulty is that the hippocampus is a complex structure. Moreover, the types of learning that involve the hippocampus are difficult to understand on a neurobiological level. The goal of the present project is to study the role of LTD in learning and memory in an animal with a simple nervous system. The hope is that the insights gained by studying the cellular mechanisms of LTD, and its role in learning, in a relatively simple organism will help us to understand the mechanisms and learning- related role of LTD in the brains of more complex organisms, including man. Toward this end the PI will study LTD in an invertebrate organism, the marine snail Aplysia californica. This animal possesses a relatively simple nervous system and exhibits several simple forms of learning. Furthermore, a great deal is already known about the neural mechanisms of Aplysia's behavior. The PI has previously shown that synapses between sensory and motor neurons in the nervous system of Aplysia can exhibit LTD. The PI will analyze the cellular mechanisms of LTD of the synapses between sensory and motor neurons in Aplysia. The PI will also determine whether LTD plays a role in a simple form of learning exhibited by Aplysia known as long-term habituation. The results of this project should provide important general insights regarding the cellular mechanisms of LTD and the role that LTD plays in learning. It is expected that these insights will advance knowledge about how nervous systems mediate learning and memory.

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.

DESCRIPTION (provided by applicant): Habituation is a basic, ubiquitous form of learning and memory. Nonetheless, the neurobiological basis of habituation in mammals, including man, is poorly understood. This situation is largely due to the vast complexity of the mammalian central nervous system. The long-term goal of this project is to use a simple, model organism, the marine snail Aplysia californica, to elucidate the cellular and molecular basis of habituation, particularly long-term habituation (LTH). When tactile stimuli are applied to the body surface of Aplysia, the animal exhibits with a defensive withdrawal reflex. In response to repeated tactile stimulation the reflex habituates. The withdrawal reflex exhibits both short- and long-term habituation. A major advantage of the withdrawal reflex for a study of habituation is that the neural circuitry that underlies the withdrawal reflex is well characterized. The proposed research will use a reduced preparation of Aplysia that can facilitate relating synaptic changes to behavioral changes. The synapse between the sensory and motor neurons that mediate the withdrawal reflex exhibits a form of long-term depression (LTD); this LTD may play a role in LTH in Aplysia. The potential role of LTD in LTH will be tested with electrophysiological and pharmacological experiments. In other experiments the cellular and molecular mechanisms of LTD will be characterized. One intriguing possibility is that LTD involves down-regulation of the function of a class of postsynaptic glutamate receptors (AMPA receptors). Functional down-regulation of AMPA receptors, perhaps due to changes in protein or mRNA synthesis, may also play a role in LTH. Pharmacological and, ultimately, molecular techniques will be used to test this idea. It is expected that the findings from the proposed research will contribute to a fundamental understanding of the processes that underlie learning and memory. Such an understanding will facilitate the development of treatments of 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.

DESCRIPTION (provided by applicant): This application is for renewal of the UCLA Training Program in Molecular and Cellular Neurobiology (MCN), first awarded in 1995. The current application updates the MCN Program. First, whereas the previous program was confined to predoctoral trainees, the present application includes a postdoctoral training component. Second, the overall intellectual focus of the program has been narrowed. This narrowing of focus is designed to take advantage of the scientific strengths of the training faculty, and is also necessitated by the significant expansion and maturation of UCLA's neuroscience community during the previous decade. Training will now focus on an area of particular strength at UCLA-the cellular and molecular mechanisms of neural plasticity, broadly conceived. The overall goals of the MCN Program remain as they were originally: to train a cadre of neuroscience researchers who will develop the next generation of therapies and prophylaxes for diseases of the brain and central nervous system. Toward this end, this proposal includes a new course in which all trainees, both pre- and postdoctoral, are introduced to clinical disorders of behavior. This course is designed to enable trainees to conceptualize the potential relevance of their research to modern psychiatry and neurology, and thereby encourage our trainees to engage in translational biomedical research;such research will facilitate the development of new treatments for brain and behavioral disorders. The MCN Program has been remarkably successful, as indicated by the high overall quality of its trainees. Furthermore, the training environment has become greatly enriched in the last decade with the hiring of new faculty members whose primary research is in areas of molecular and cellular neurobiology. The MCN Program has facilitated the development of faculty collaborations across departmental and disciplinary boundaries. We anticipate that such collaborations, enhanced by the additional of postdoctoral trainees to the Program, will accelerate during the next five years. The Training Program will enhance understanding of the cellular and molecular bases of such diseases as schizophrenia, bipolar disorder, anxiety, autism and Alzheimer's disease. It is expected that this knowledge, together with the clinical training of the trainees, will lead to more effective treatments for these disorders.

[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]

The purpose of this project will be to develop the zebrafish (Danio rerio) into a useful model system for understanding the cellular and molecular mechanisms underlying learning and memory. Towards this end, behavioral investigations of several simple forms of learning in this vertebrate organism will be examined. The behavioral experiments will focus on experience-dependent modulation of the fish's startle reflex. The research will focus on simple forms of learning, including habituation and sensitization of the startle reflex in larval zebrafish The Principal Investigator will attempt to demonstrate more long-lasting forms of learning in zebrafish larvae, particularly intermediate- and long-term habituation and sensitization. In addition, the project will investigate whether the startle reflex can undergo associative learning. He will attempt to show a type of learned fear in the fish: the fear-potentiated startle reflex. Given that robust forms of learning can be developed for use in zebrafish larvae, pharmacological techniques will be used to begin to understand the neural and molecular processes that underlie associative learning in the fish. For example, training will be performed in inhibitors of protein or RNA synthesis in order to determine whether the various forms of memory depend on translation or gene transcription. Furthermore, specific inhibitors of the activity of various protein kinases and protein phosphatases will be used to test for roles for these kinases/phosphatases in learning in zebrafish. The proposed experiments will contribute significantly to an understanding of how the brain mediates learning and memory, as well cognition. Such knowledge should be highly useful for improving educational methods in the United States and will offer undergraduate students unique training opportunities using state-of-the-art methodologies.

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.

Historically, many of the most important advances in cellular neurobiology have been made studying mollusks, such as squid and snails, as model systems because mollusks have exceptionally large nerve cells. The giant nerve cells of squid enabled the analysis of the mechanism of the action potentials found in nerve cells and the mechanisms by which neurotransmitters, such as serotonin, are released at synapses. Snails, with their very large nerve cells and simple neural circuitry, have enabled major advances in our
understanding of molecular mechanisms of learning and memory. Many key cellular and molecular discoveries in mollusks have proved generalizable to the brains of mammals, because these mechanisms have been conserved throughout evolution.

This meeting will bring together neurobiologists who work on two important molluscan groups, the simpler gastropods (e.g. snails) and the more complex cephalopods (e.g.squid and octopus) to freely discuss their latest findings. Octopus and squid represent advanced forms of intelligence that evolved entirely independently from vertebrates. These distinct forms of intelligence have the potential for providing influential models for developing artificial intelligence and computer-controlled precise motion of artificial appendages.

The meeting will generate novel collaborations among attendees, particularly because this is a unique venue for discussions between leaders in gastropod and cephalopod neuroscience. Cellular, molecular and genomic advances in gastropods have the potential for greatly benefiting research in more complex cephalopods. Abstracts of talks will be available on an open website, along with new methodologies presented. This will accelerate the pace of research, leading to new discoveries that can provide insights into the basic functioning of the brain. Graduate students and postdoctoral fellows will present their research to senior scientists in the field, and interact with them at mentoring sessions, which will benefit their development as researchers.

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.

One of the most important problems in neurobiology is how memories are maintained in the brain over the course of decades. Traditionally, it has been thought that, after induction and an initial period of consolidation, memories are relatively permanent and resistant to disruption. Recent evidence, however, indicates that long-term memories are far more labile than previously supposed. Evidence for the lability of long-term memory has come from studies in which a consolidated memory is reactivated in an animal, typically by giving the animal a stimulus that reminds it of the original learning experience. It has been found that reactivation renders a stored, long-term memory subject to disruption by manipulations, such as inhibition of protein synthesis, that disrupt newly formed memories. This finding has led to the idea that, upon reactivation, long-term memories undergo a new episode of consolidation ("reconsolidation"), during which they are fragile and subject to elimination. Although the notion that consolidated memories undergo reconsolidation following their reactivation is increasingly accepted by neurobiologists, the mechanisms of memory reconsolidation are poorly understood. This project will use a simple invertebrate organism to gain general insights into the cellular and molecular processes that mediate memory reconsolidation. A significant advantage of this model system is that memory reconsolidation can be examined at the level of a single sensorimotor synapse in dissociated cell culture. This permits rigorous mechanistic analyses of reconsolidation. Among the questions that will be addressed during the project are the extent to which disruption of reconsolidation of a memory actually eliminates the memory; the similarity between the molecular signaling processes that are involved in original memory consolidation to those involved in memory reconsolidation; and the effect of reconsolidation on the strength of the memory. The latter question will be examined on both the behavioral and synaptic levels. The broader impacts of this project include the training of underrepresented graduate students and undergraduates. In addition, significant public outreach will be undertaken.