Nicholas C. Spitzer - US grants

This project will provide information about timing of RNA and protein syntheses necessary for the expression of specific neuronal phenotypes during development. We have described the sequence of appearance of some cellular properties of amphibian spinal neurons differentiating in vivo. The same developmental changes occur in embryonic spinal neurons dissociated and grown in culture. Specific metabolic inhibitors will be added to the culture media at later and later stages of development, and the time of application at which their continuous presence no longer blocks expression of electrical excitability, chemosensitivity and high affinity neurotransmitter uptake will be determined. Brief applications of reversible metabolic inhibitors will provide information about the existence of critical periods, during which expression of single phenotypes may be blocked. If such critical periods exist, it will be possible to examine the consequences of the absence of one phenotype on the expression of others.

Answers to the questions posed in this proposal would offer insights to one of the most difficult and intriguing of developmental processes, the formation of neural connections in the embryonic central nervous system of vertebrates. Two specific issues are addressed: (1) How are nerve fibers guided to their proper targets in the developing central nervous system? (2) What factors influence the formation of functional connections between these processes and their target cells? The majority of the experiments described use embryonic surgery on amphibians, different genotypes, or different embryonic stages will be used to explicitly test proposed roles for (1) diffusable factors, (2) neural impulse activity, (3) timing. Results from these experiments will be further pursued by asking (1) whether cues that are used in the formation of neural pathways are common to different vertebrate species, (2) if the elimination of any single cue does not interfere with axonal navigation whether the elimination of combinations of cues does so, and (3) whether we can demonstrate axonal navigation to a target in vitro. A culture system with time-lapse video recording will also be used to assay turning responses to retinal axons to factors exuded by or extracted from target tissues. The long term goal of this research would be to identify specific biological factors and their roles in neural development. Such knowledge would allow us to focus attention on these factors individually and finally come to a cellular and molecular understanding of the formation of neural connections. Such knowledge might also be of significant medical value with respect to the prevention and treatment of malformation in human fetal brain.

DESCRIPTION: (Applicant's Abstract) Our previous work shows that embryonic amphibian spinal neurons exhibit spontaneous elevations of intracellular Ca2+ at early stages of development prior to synapse formation both in dissociated cell culture and in vivo. Further, our recent results indicate that these early Ca2+ transients specify key aspects of neuronal differentiation in a frequency encoded manner. These findings suggest that Ca2+ elevations initiate signal transduction cascades that determine subsequent steps of development. The proposed research has four specific aims. The 1st and 2nd Aims investigate the molecular regulation of neuronal phenotypes that are specified by spontaneous elevations of intracellular Ca2+. The 3rd and 4th Aims test hypotheses regarding regulation of differentiation by oscillations of second messengers, either at different developmental stages or via a different second messenger (cyclic AMP). The first aim investigates the hypothesis that particular frequencies of spontaneous Ca2+ spikes specify the cholinergic phenotype in spinal neurons, both in culture and in vivo. The second aim investigates the function of Ca2+ waves in axonal pathfinding in vivo, and analyzes the signal transduction cascade by which Ca2+ waves affect specific components of the growth cone cytoskeleton. The third aim is to investigate the role of Ca2+ transients in primary induction, which occurs well before terminal differentiation of neurons. The fourth aim is to investigate the interaction between elevations of cAMP and Ca2+ and the impact of this interaction on neuronal differentiation, and to analyze the incidence of spontaneous elevations of cAMP in embryonic neurons. A combination of embryological, imaging, electrophysiological, immunocytochemical and molecular biological techniques will be used to fulfill these aims. The immediate goal is to test hypotheses about specific mechanisms underlying differentiation of vertebrate spinal neurons in order to define the roles of transient elevations of second messengers in driving differentiation. The long term goal is to provide information about the cellular and molecular machinery that governs processes of development. It is expected that this work will contribute to an understanding of developmental disorders of the nervous system.

Lineage studies have shown that retinal cells, as well as many other cells in the vertebrate nervous system, are born pluripotent Culture studies have demonstrated that cellular interactions can drive these pluripotent cells toward particular fates. This is a proposal to investigate the nature of these inductive pathways. For Xenopus photoreceptors, there are two temporally distinct inductive events. The first is necessary to turn on some antigens expressed in both rods and cones, and the second one induces cells to express rod specific markers. Two general schemes of determination are consistent with these separate inductions. In one a single cell is exposed to a series of inductive events that increasingly restrict its fate; in the other, single distinct interactions induce the different cell types. One aim of this proposal is to distinguish between these two possibilities by a using a combination of mixed cell cultures and immunocytochemical tagging. Another main objective of this proposal is to test explicitly whether similar inductive schemes are used for other cell types in the retina. There is an evolutionarily conserved order in retinal histogenesis in vertebrates, and we will investigate the possibilities that this occurs because inductive signals arise in a particular sequence, or because cells change their competence to respond to inductive signals. Changes in competence will be assayed by removing labeled cells from the retina at different stages of development, exposing them to the same inductive cues, and examining the appearance of particular cell types among the labeled cells. The last major goal of this proposal is to examine the role of particular growth factors in retinal development in vivo, by misexpression of dominant-activated and dominant- negative forms of growth factor receptors m vivo, and examining retinal cell development immunohistochemically. The long term objectives of this proposal are to understand the cascade of cell determination in the vertebrate nervous system at a cellular and a molecular level. One has to understand who induces who, what the signals are, and what the receptors are. This proposal paves the way for a better understanding of the order of cellular genesis in the nervous system, in particular a working model for the generation of organized cellular complexity from a specific sequence of inductive events. The retina is a highly accessible and well studied part of the vertebrate central nervous system and will serve well as an experimental system for future work of this sort. The basic understanding of how cells are determined in the retina, and the capability of controlling these inductive events in culture and in vivo, may have implications for retinal regeneration after surgery or injury. This work may also have applications in understanding the embryonic malformations of the retina.

DESCRIPTION (provided by applicant): This program has the purpose of preparing postdoctoral students for careers in biomedical research and teaching. Fields of specialization are developmental neurobiology, integrative neurophysiology and small systems analysis, systems neuroscience, biophysics, molecular neurobiology, neurogenetics, neuroanatomy and neurochemistry. The form of the training further illustrates the interdisciplinary nature of the program. Primary emphasis is given to research undertaken through cooperation of several neurobiology laboratories, each with its own specific approach. Training takes place in the Division of Biological Sciences of UCSD which fosters a spirit of interdisciplinary fusion motivated by the founders of the campus, in which many different types of biology are being pursued and many different approaches to solving problems are being shared. All participants have access to the wider neurobiological community, including the faculty and equipment at institutions affiliated with UCSD: The Neuroscience Department of UCSD's School of Medicine, the Howard Hughes Medical Institute, the Scripps Institution of Oceanography, the Salk Institute, the Scripps Research Foundation and the Neurosciences Institute. These resources have proved to be valuable components of the training environment. Seminars include weekly journal clubs, weekly talks from local and visiting neurobiologists, and specialized courses on a wide range of topics. These complement the research program, which is conducted under the sponsorship of individual faculty members. Postdoctoral trainees also participate in the teaching done by faculty, by lecturing in specialized neurobiology courses. However emphasis is placed on developing the potential of individual trainees for independent and original research. The four postdoctoral trainees are typically Ph.D.s, usually from other neurobiology programs, although they occasionally come from other areas of science. The selection criteria are superior scholarship in rigorous undergraduate and graduate programs, solid evidence of excellent and original research abilities, recommendations from past advisors, and expressions of commitment to careers in biomedical research. Effort is made to identify individuals of minority status. Research training takes place chiefly in the laboratories of the participating faculty, although the close ties with other laboratories and institutes and the interdisciplinary spirit of the community make it likely that some parts of a trainee's research will take place in other local laboratories.

DESCRIPTION (modified from applicant's abstract) This project focuses on spontaneous elevations of intracellular calcium in growth cones (calcium waves) that regulate extension of neurites. The outgrowth of neurites to establish specific neuronal connections is an important aspect of early development of the nervous system. The research addresses the mechanisms of generation of calcium waves in growth cones of amphibian spinal neurons in culture and has two major specific aims. The first aim is to investigate the contributions of a potentially novel calcium leak pathway in combination with known calcium transporters to the generation of calcium waves. The second aim is to determine the identify and roles of intracellular calcium stores in amplifying elevations of intracellular calcium in growth cones and thereby regulating neurite extension. Optical imaging of intracellular calcium and recording of ionic currents will be used to address these aims.

DESCRIPTION (provided by applicant): In previous work we discovered three classes of spontaneous transient elevations of intracellular calcium (Ca) in embryonic Xenopus spinal neurons and showed that they have distinct functions, regulating aspects of differentiation in a frequency-dependent manner prior to synapse formation: 1) Ca spikes are generated by developmental transient Ca-dependent action potentials and regulate expression of neurotransmitters. 2) Growth cone Ca transients are generated locally in the growth cone and regulate the rate of axon extension. 3) Filopodial Ca transients are produced at the tips of filopodia and regulate growth cone turning. The proposed research has three specific aims that address the mechanisms of action of Ca transients in differentiation of these embryonic spinal neurons. The first aim investigates the mechanisms by which Ca spikes regulate neurotransmitter expression. The second aim analyzes the mechanism generating changes in membrane potential that lead to onset and termination of Ca spiking. The third aim investigates the mechanism by which voltage-gated Ca channels regulate growth cone function. We describe experiments to address the following questions: What molecules are involved in the action of Ca spikes? What mechanisms generate the changes in membrane potential that lead to onset and termination of the period of Ca spiking? Does the mechanism of action of growth cone voltage-gated Ca channels involve interaction with extracellular matrix molecules or mechanically enhanced transmitter release? The immediate goal of this research is to test hypotheses about the mechanisms of Ca transients in the early stages of differentiation of vertebrate spinal neurons. The long term goal is to provide information about the cellular and molecular machinery that governs processes of development, which will contribute to understanding developmental disorders of the nervous system.

DESCRIPTION (provided by applicant): Our previous work discovered developmental changes in expression of voltage-dependent and ligand-activated channels during early differentiation of amphibian spinal neurons. These findings raised the possibility that early forms of ion channel activity participate in signal transduction that influences subsequent steps of development. In recent studies we have discovered that spontaneous calcium spikes regulate expression of neurotransmitters in neurons of the spinal cord, identified ligands that drive this spontaneous activity, and shown that neurons with a modified transmitter phenotype select cognate receptors and form functional synapses. These findings lead to several related questions: 1) How general is activity-dependent transmitter specification, i.e. does calcium spike activity also regulate transmitter expression in the brain, and if so what are the underlying mechanisms? 2) Do natural environmental stimuli such as ambient light or temperature also influence transmitter specification, i.e. does the global environment in which differentiation occurs modulate this developmental process? The experimental perturbations previously exploited are not ones encountered during normal development and leave open the question of whether natural environmental stimuli influence the choice of transmitter for different classes of neurons. 3) Are there local environmental factors that interact with spike activity and contribute to transmitter specification? Our proposed research has four specific aims that analyze activity-dependent expression of biogenic amines (dopamine and serotonin) in the brain (aims 1 &2), address the function of natural environmental stimuli in specification of transmitter expression (aims 1-3), and analyze several regulatory mechanisms (aims 2 &4). The 2nd aim analyzes the role of activity-dependent transcription factors in regulating transmitter expression and the 4th aim investigates the interaction between early electrical activity and target-derived factors in transmitter specification. The immediate goal of this research is to test hypotheses about the role of electrical activity and natural environmental stimuli in transmitter specification in the vertebrate CNS. The long term goal is to provide information about the cellular and molecular machinery that governs neurotransmitter specification during development. It is expected that this work will contribute to understanding developmental disorders of the nervous system in which neurotransmitter expression is altered during embryonic and post-embryonic development. PUBLIC HEALTH RELEVANCE: Cognitive disorders such as depression, schizophrenia and Parkinson's disease are disorders of neurotransmitter and neurotransmitter receptor metabolism. This research will provide information about the role of electrical activity and natural environmental stimuli in transmitter specification in the vertebrate nervous system. It is expected that this work will contribute to understanding developmental disorders of the nervous system in which neurotransmitter expression is altered during embryonic and post-embryonic development.

DESCRIPTION (provided by applicant): In previous work we demonstrated that Ca spikes generated by developmentally transient Ca-dependent action potentials regulate expression of neurotransmitters. We showed that this regulation was homeostatic, such that an increase in the Ca spike activity resulted in an increase in the incidence of cells expressing inhibitory neurotransmitters (glycine and GABA) and a concomitant decrease in the incidence of excitatory neurotransmitters (glutamate and acetylcholine). Conversely, a decrease in Ca spike activity caused an increase in the incidence of excitatory neurotransmitters and a decrease in inhibitory neurotransmitters. The proposed research has two specific aims targeted at generating mutant and transgenic lines in Xenopus tropicalis useful for identifying molecules involved in the Ca spike-dependent specification of neurotransmitters and for studying activity-dependent development in general. The first specific aim uses a gain-of-function screen and the second specific aim uses gene trap insertional mutagenesis. The third specific aim characterizes a subset of the genes identified in the first and second aims. The immediate goal of this research is to identify molecules involved in Ca spike- dependent neurotransmitter specification and to generate reagents for further research in this area. In addition, we aim to generate information and mutant lines that are of value to the Xenopus community. The long-term goal is to provide information about the molecular signaling pathways that govern processes of neuronal development, which will contribute to understanding developmental disorders of the nervous system. PUBLIC HEALTH RELEVANCE: Correct specification of neurotransmitter expression during embryonic development is essential for the function of the nervous system, and both gene expression and electrical activity contribute to this process. However the mechanisms by which nature (genes) and nurture (activity) interact to specify the appropriate expression of neurotransmitters are unclear. This research will identify activity-dependent genes regulating transmitter expression. It is expected that this work will contribute to understanding developmental disorders of the nervous system in which neurotransmitter expression is altered during embryonic and post-embryonic development.

DESCRIPTION (provided by applicant): Project Abstract The Neural Circuits Postdoctoral Training Program at UC San Diego is in its 30th year. It is central to a vibrant neuroscience community of research and training in the Neurobiology Section. 20 laboratories focused on different aspects of the development, structure, function and plasticity of neural circuits work together with the goal of preparing postdoctoral fellows for careers in biomedical research and teaching. The Program Director is Nicholas C. Spitzer, Vice Chair of Neurobiology, Professor of Biological Sciences and Co-Director of the Kavli Institute for Brain and Mind. The Executive Committee consists of four faculty who serve staggered three year terms (currently Profs Anirvan Ghosh, Massimo Scanziani, Yimin Zou and Stefan Leutgeb). Training takes place in the Division of Biological Sciences that fosters a spirit of interdisciplinary fusion motivated by the founders of the campus, in which many different types of biology are being pursued and many different approaches to solving problems are being shared. Primary emphasis is placed on developing the potential of individual trainees for independent and original research. However trainees acquire broad knowledge of neural circuits teaching courses and attending seminars. All participants have access to the wider neurobiological community, including the faculty and equipment at institutions affiliated with UCSD: The Neuroscience Department of UCSD's School of Medicine, the Salk Institute, the Scripps Research Institute, the Scripps Institution of Oceanography, and the Neurosciences Institute. These resources have proved to be valuable components of the training environment. Seminars include weekly talks on a wide range of topics from local and visiting neurobiologists at all of these institutions. These complement the research program, which is conducted under the sponsorship of individual faculty members. The Neural Circuits Training Program provides outstanding fellows with an identity, and promotes resourcefulness and an atmosphere of commonly shared inquisitiveness in a rich and collaborative environment. Support for 4 postdoctoral positions at 02-04 levels is requested (out of 68 postdoctoral fellows currently in participating laboratories). Trainees are typically Ph.D.s from other neurobiology programs. The selection criteria are superior scholarship in rigorous undergraduate and graduate programs, solid evidence of excellent and original research abilities, recommendations from past advisors and expressions of commitment to careers in biomedical research. Particular effort is made to identify individuals of minority status. Trainees are supported in their first year, during which they apply for their own support for subsequent years, and participate in the program for three years.

DESCRIPTION (provided by applicant): The function of the nervous system depends on the appropriate specification of neurotransmitters and their receptors that enable the function of neural circuits. We have discovered that circuit activity plays a key role in transmitter specification (Dulcis and Spitzer, 2008; Dulcis et al., 2013) and that changes in transmitter expression are matched by corresponding changes in postsynaptic receptors (Borodinsky & Spitzer, 2007; Dulcis & Spitzer, 2008; Dulcis et al., 2013), causing changes in behaviors. We now propose to determine whether neurotransmitter switching can be induced by patterns of neuronal activity that induce LTP in the adult rat hippocampus, and whether this is accompanied by changes receptor population. We will also determine whether environmental enrichment and fear conditioning that engage the hippocampus induce neurotransmitter switching. We will test the role of newly expressed transmitters in adult neurogenesis and in the development of resilience to acute and chronic stress. The immediate goal of this research is to test the hypothesis that activation of neural circuits in the hippocampus leads to transmitter respecification in neurons in these circuits, with corresponding changes in adult neurogenesis and/or stress behavior. A long-term goal is to understand how neural network activation can be used to induce neurotransmitter plasticity in the adult brain and ultimately to advance the mission of the NIH to treat neurological disorders.

DESCRIPTION (provided by applicant): Electrical activity and calcium signaling have significant roles in directing neurotransmitter specification. Changes in electrical activity by natural visual stimuli trigger the appearance of neurotransmitters in neurons that normally produce different ones. Many of studies of these processes have been carried out in cell culture, providing important insights into the capabilities of developing neurons. Ultimately, however, we want to understand how differentiation proceeds in vivo. The proposed research has three specific aims, focused on understanding these processes in the intact nervous system of larvae of the frog, Xenopus laevis, which facilitates behavioral, cellular and molecular analysis. The immediate goal of this research is to test the hypothesis that activation of a neural circuit by a natural olfactory stimulus leads to transmitter respecification in neurons in the circuit with corresponding changes in behavior controlled by the activated circuit, and that specific molecular mechanisms are involved. The long- term goal of this research is to understand how neural network activation can be used to induce neurotransmitter plasticity in the brain.

Project Summary Abstract: Repeated exposure to a drug of abuse can lead to the development of an addiction, a chronic and relapsing illness that has been defined as the compulsion to seek and take the drug, the loss of control in limiting intake, and the emergence of a negative emotional state. The transition from drug intake to addiction involves neuroplasticity of brain circuits related to reward, impulse control and affect. One of the brain regions that is functionally impaired by drug intake is the prefrontal cortex (PFC), in which drugs of abuse induce alterations in activity that seem to be crucially involved in the loss of control over drug intake. Increasing our knowledge of the ways in which drugs of abuse impact these circuits is important to better understand the mechanisms underlying the transition to addiction and to allow the development of novel interventions and treatment. The proper function of brain circuits relies on the correct specification and orchestration of neurotransmission. Accumulating evidence has recently established that when prolonged environmental stimuli produce a sustained alteration in the electrical activity of a subset of neurons, these neurons can undergo a respecification of the neurotransmitter they express, often resulting in a switch from an excitatory to an inhibitory transmitter or vice versa. This newly appreciated form of neuroplasticity, called neurotransmitter switching, has been repeatedly observed in the adult mammalian brain and associated with alterations in animal behavior. We hypothesize that repeated exposure to drugs of abuse can induce neurotransmitter switching that in turn contributes to the establishment of drug-induced behavioral alterations such as cognitive deficits, behavioral sensitization and drug-induced reinforcement. Two specific aims will test this hypothesis. The 1st specific aim will test whether sub-chronic treatment with PCP or METH induces neurotransmitter switching in the PFC. The 2nd specific aim will determine whether there is a causal link between neurotransmitter switching and drug- induced behavioral alterations including cognitive deficits, behavioral sensitization, and drug-induced reinforcement.

Project Summary Abstract: Our perceptions, behaviors, emotions, memories and intelligence depend on the appropriate synthesis and release of specific neurotransmitters in the brain. Transmitter identity is initially established by genetic programs. It has been thought that transmitters are fixed and invariant throughout life and that the plasticity of the nervous system consists largely of changes in the strength and number of synapses. We have found that experimental perturbations of spontaneous electrical activity and natural changes in sensory stimuli such as ambient light or odors respecify transmitter identity in the spinal cord and brain in the developing nervous system, leading to matching changes in postsynaptic transmitter receptor specification and changes in animal behavior. Strikingly we recently found that transmitter switching and receptor matching also occur in the adult mammalian brain in response to sensory stimuli and can regulate behavior. These discoveries contrast sharply with the general view of transmitter constancy and identify another way that the nervous system adapts to the environment. Here we describe experiments to determine the effect of motor activity in driving transmitter respecification causing changes in behavior. Our proposed research has three specific aims. The 1st aim tests the hypothesis that sustained running activity leads to enhanced acquisition of other motor skills. The 2nd aim tests the hypothesis that sustained running causes the loss of one transmitter that is accompanied by the gain of another transmitter. The 3rd aim tests the hypothesis that preventing the changes in transmitter identity prevents the changes in behavior that are generated by motor activity. The immediate goals of this research are to test specific hypotheses about the effect of motor activity in generating a novel form of plasticity that involves changes in transmitter identity in the adult mammalian brain. We will determine the role of this form of plasticity in the changes in locomotor behavior. The long-term goals are to understand the role of neurotransmitter respecification in procedural learning and memory.

Project Summary Abstract: Repeated exposure to a drug of abuse induces alterations in neuronal activity and neuroplastic changes in multiple brain areas and circuits related to reward, impulse control and affect. These widespread changes contribute to multiple behavioral alterations. Despite differing in their molecular targets, different drugs cause common alterations in the brain. Increasing our knowledge of the impact of drugs of abuse on multiple brain regions and circuits is crucial to better understand the mechanisms that mediate the transition from drug use to addiction and facilitate the development of novel treatment. We hypothesize that phencyclidine induces neurotransmitter switching in more than just one brain region and that different categories of drugs induce a shared transmitter switch that is relevant for the transition from drug intake to addiction. To test these hypotheses we are developing genetic mouse models to facilitate high- throughput, whole-brain screening of transmitter switching involving GABA and glutamate. The current assays of transmitter switching involve time consuming sectioning of a single parts of the brain, staining sections and stereology, putting whole-brain analysis out of reach. The combination of genetically encoded fluorescent reporter proteins, brain clarification, light sheet microscopy and whole-brain automatic image analysis will speed analysis and accelerate understanding of the role of transmitter switching in drug-induced neuroplasticity and drug-induced behavioral alteration. These tools will be used to determine whether two different substances of abuse that have distinct targets in the brain, PCP and methamphetamine (METH), induce NTS in multiple brain regions and whether some PCP- and METH-induced NTS are the same.

Project Summary Abstract: Our perceptions, behaviors, emotions, memories and intelligence depend on the appropriate synthesis and release of specific neurotransmitters in the brain. Transmitter identity is initially established by genetic programs. It has been thought that transmitters are fixed and invariant throughout life and that the plasticity of the nervous system consists largely of changes in the strength and number of synapses. We have found that experimental perturbations of spontaneous electrical activity and natural changes in sustained sensory stimuli such as ambient light or odors respecify transmitter identity in the spinal cord and brain in the developing nervous system, leading to matching changes in postsynaptic transmitter receptor specification and changes in animal behavior. Strikingly we found that transmitter switching and receptor matching also occur in the adult mammalian brain in response to sustained sensory stimuli and can regulate behavior. These discoveries contrast sharply with the general view of transmitter constancy and identify another way that the nervous system adapts to the environment. Here we describe experiments to determine how many transmitter switches are induced by a single environmental stimulus and how many brain regions are affected. There is increasing understanding that the brain is a widely linked network and that single perturbations alter activity throughout the brain. It is important to address this issue in order to understand better the basis of changes in behavior in response to the sustained stimuli that are major determinants of our conduct. A major part of our behavioral and cognitive repertoire is habitual and results from sustained experience. We will also analyze the mechanisms that promote and modulate transmitter switching. Although it is clear that neurotransmitter switching is activity- dependent, the features of activity that are necessary to achieve switching remain unknown. In the future this knowledge may have clinical utility for driving or preventing transmitter switching in patients. The immediate goals of this research are to test specific hypotheses about the effect of activity in generating a novel form of plasticity that involves changes in transmitter identity in the adult mammalian brain. The long- term goals are to understand the role of neurotransmitter switching in regulating behaviors.