Mu-Ming Poo - US grants

This is a five-year research plan to study the molecular and cellular events associated with (1) the growth and orientation of nerve processes, (2) the interaction between nerve and muscle cells before and shortly after their physical contact, and (3) development of the neuromuscular synapse. Specifically, we will examine the release of molecules from both the nerve and muscle cells, the response of growing neurite to a gradient of chemotropic substances, quantal and non-quantal components of transmitter release from neurons before and after nerve-muscle contact, and the role of synaptic activity in maturation of the synapse and in modulating the growth and stabilization of nerve terminals. These problems will be studied in nerve-muscle cultures prepared from the Xenopus embryos, using a combination of electrophysiological and cell biological techniques, including a newly-developed method of an excised membrane patch for assaying minute quantities of channel-inducing substances, and a whole-cell voltage-clamp method for recording synaptic currents. These studies will not only provide us new information concerning various cellular events during early phases of synaptogenesis, but are also likely to reveal some of the cellular mechanisms underlying the guidance of nerve growth, release of transmitter at the nerve terminal, and plasticity of the developing neuromuscular synapse. Our long-range goal is to understand the molecular and cellular mechanisms responsible for the development of specific neuronal connections.

Cell surface interaction during the early phase of nerve-muscle contact is of crucial importance to the development of the neuromuscular synapse. This research will center around the molecular mechanisms underlying two early events during the initial nerve-muscle contact, namely, the contact-dependent induction of acetylcholine release from the embryonic Xenopus spinal neuron and the development of nerve-muscle adhesion. It will be determined whether muscle surface acetylcholine receptors and other cell surface components are involved in these two events. It will also be determined whether there is specificity for nerve-muscle interaction among subpopulations of spinal neurons and myotomal muscle cells that depends upon the axial position or the lineage of the cells in the embryo. Finally, the study will focus on what effect the interfering of these two early events will have upon the structural and functional development of the synapse. Results from these studies will help us to understand the cellular and molecular mechanisms underlying the development of specific neuronal connections.

The function of the nervous systems depends on the signaling of nerve impulses across the nerve cells at a structure called synapse. A group of molecules in the membrane of the receiving post-synaptic nerve cells, the transmitter receptors, are responsible for receiving the chemical signals from the pre- synaptic nerve cells. The metabolism, mobility, and the distribution of these receptors determine the efficiency and stability of the signaling at the synapse. The purpose of the present project is to examine when the glutamate receptors are synthesized in the developing nerve cells, how do they become segregated into different regions of the cell membrane, how their distribution at the synapse become concentrated as synapse matures, and whether the concentrated distribution can be further modified by the use of the synapse. The result from this study will help us to understand the role of transmitter receptor dynamics in the synaptic functions as well as in the disease states, during embryonic development and in adult life.

This is a five-year plan to continue our research on the molecular and cellular events associated with (1) the growth and orientation of nerve processes, (2) the secretion of acetylcholine (ACh) and other factors from the nerve terminal before and after the contact with its target muscle cell, (3) the early nerve-muscle interactions and the maturation of neuromuscular synapse. These events will be studied in Xenopus nerve- muscle cultures, using a combination of techniques from electrophysiology, biophysics, and cell biology. We will address problems associated with three different stages of neural development. First, for the period of nerve growth, we will examine the effect of neuronal activity on neurite extension, the relationship between neurite extension and transmitter secretion, and the role of cytoplasmic second messengers, Ca2+ and cAMP in particular, in regulating the secretion and orientation at the growth cone. Second, for the early period of synaptogenesis, we will study the neuronal responses to muscle contact, the involvement of ACh receptors in nerve- muscle interactions, and the secretion of calcitonin gene-related peptide from the nerve terminal. Third, for the period of synapse maturation, we will survey the structural and functional changes associated with synaptic maturation, examine the role of second messenger systems in regulating these changes, and test the hypothesis that synaptic activity affects the development and stability of the synapse through direct electrokinetic actions exerted by the synaptic currents on the distribution of cellular components. The result of this research will help us to understand the molecular aspects of nerve growth, transmitter secretion and formation of synaptic connections.

One of the most significant advances in our understanding of development of the brain during the past two decades is the realization that nerve connections made during embryonic life undergo substantial rearrangement as the nervous system matures. Some connections are eliminated, while others become strengthened and stabilized. Evidence suggests that this rearrangement results from a competitive interaction between adjacent nerve connections in a manner that depends upon the pattern of nerve activity, which is in turn triggered by the sensory input from the environment. This activity-dependent competition between adjacent connections has been observed in both central and peripheral nervous systems, but the mechanism responsible for this phenomenon is poorly understood. In the previous granting period of this project, it was discovered that activity-dependent competition can be observed in isolated synaptic connections in a simple cell culture. This finding opens the possibility of experimentally addressing the problem of synaptic competition effectively at cellular and molecular levels. In the present project, a series of experiments will be carried out that will result in an insight into the mechanism responsible for modulating the strength of synaptic connections. This should lead to an understanding of how adjacent synapses on a single postsynaptic cell compete for survival and of the physiological and morphological changes at the synapse during the process of synaptic competition. The results from these studies will help us to understand how nerve connections are modified during development of the nervous system.

This is a five-year plan to continue our research on the molecular and cellular events associated with (1) the growth and orientation of nerve processes, (2) the secretion of acetylcholine (ACh) and other factors from the nerve terminal before and after the contact with its target muscle cell, (3) the early nerve-muscle interactions and the maturation of neuromuscular synapse. These events will be studied in Xenopus nerve- muscle cultures, using a combination of techniques from electrophysiology, biophysics, and cell biology. We will address problems associated with three different stages of neural development. First, for the period of nerve growth, we will examine the effect of neuronal activity on neurite extension, the relationship between neurite extension and transmitter secretion, and the role of cytoplasmic second messengers, Ca2+ and cAMP in particular, in regulating the secretion and orientation at the growth cone. Second, for the early period of synaptogenesis, we will study the neuronal responses to muscle contact, the involvement of ACh receptors in nerve- muscle interactions, and the secretion of calcitonin gene-related peptide from the nerve terminal. Third, for the period of synapse maturation, we will survey the structural and functional changes associated with synaptic maturation, examine the role of second messenger systems in regulating these changes, and test the hypothesis that synaptic activity affects the development and stability of the synapse through direct electrokinetic actions exerted by the synaptic currents on the distribution of cellular components. The result of this research will help us to understand the molecular aspects of nerve growth, transmitter secretion and formation of synaptic connections.

DESCRIPTION (Adapted from applicant's abstract) : This is an application to continue studies on the cellular/molecular mechanisms underlying neuronal growth responses to extracellular signals, the development and maintenance of distinct plasma membrane domains in neurons, and the retrograde influence of postsynaptic muscle cells on the differentiation and functioning of presynaptic neurons during the early phase of synaptogenesis. The studies are focused on the spatial and temporal properties of cytosolic signaling mechanisms and the regulation of membrane trafficking in developing neurons. Cultured neurons and myotubes from Xenopus and neurons from rat hippocampi will be used. The specific aims are to understand 1) the role of Ca2+ and cAMP as second messengers in regulating the rate and direction of neurite extension, 2) early cellular events leading to the turning of growth cones induced by diffusible chemotropic substances, 3) mechanisms by which new membrane material is incorporated into the plasmalemma, 4) mechanisms underlying the formation and maintenance of distinct axonal vs. somatodendritic plasma membrane domains in hippocampal pyramidal neurons, 5) influence of muscle contact on the motility and morphology of growth cones, the localization of plasma membrane proteins, and the trafficking of cytoplasmic organelles, and 6) long-range signaling within the neuronal cytoplasm triggered by local synaptic contacts and trans-synaptic retrograde signals, and consequences on transmitter secretion from other parts of the neuron. Using a combination of electrophysiological, optical, and molecular techniques, including patch-clamp electrophysiology, Ca2+ imaging, photoactivation of caged compounds, and expression of chimeric green fluorescent proteins, the proposed studies will yield novel information concerning the mechanisms responsible for regulating the growth and synaptogenesis of developing neurons.

This proposal aims to elucidate the molecular nature of growth cone guidance and target recognition in the developing visual projection of the vertebrate, Xenopus laevis. A multidisciplinary approach is proposed to address questions in three main areas. First, the role of a polypeptide growth factor, basic fibroblast growth factor (bFGF), will be examined based on our recent finding that bFGF, when added exogenously to living brains, causes the axons of retinal ganglion cells to by-pass, rather than innervate, their target the optic tectum. The effects of bFGF on retinal axon growth will be assayed in culture and in vivo with timelapse videomicroscopy and the normal expression pattern of bFGF, and the FGF receptor (FGFR), will be characterized with immunocytochemistry. The functional roles of bFGF and FGFR signaling will be tested by a) the introduction of dominant negative, constitutively activated and inducible forms of the FGFR into retinal ganglion cells in vivo; and b) the addition of synthetic peptides that block bFGF-FGFR interactions to the developing pathway. Second, the biological roles of two glycosaminoglycans, heparin and chondroitin sulfate (CS) will be characterized following our observations that heparin, like bFGF, causes retinal axons to by-pass the tectum whereas CS abolishes pathfinding. The similarity of the mistargeting phenotypes induced by exogenous heparin and bFGF, together with the fact that heparin/heparan sulfate is a required co-factor for FGF/FGFR interactions, suggests that the action of heparin is mediated via the bFGF or FGFR signaling pathways. Experiments are proposed to test this idea. The in vivo role of CS will be investigated with immunolocalization, enzymatic degradation and binding studies, and an in vitro substrate choice assay system will be used to address how CS might modulate growth cone steering. Third, the question of when the neuroepithelium first develops patterned cues that retinal axons use to navigate by will be addressed with transplants of embryonic brain tissue. Retinal ganglion cell axons will be challenged with increasingly younger pieces of optic tract and tectum to determine when pathway guidance and target recognition cues first arise. The experiments here focus primarily on in vivo development with the goal of identifying the molecular processes that are biologically relevant. The possibility that growth factors and glycosaminoglycans play fundamental roles in axon guidance and target recognition in the developing retinal projection has not been examined previously and our proposed studies promise to yield novel insights into this area.

DESCRIPTION: Target-derived neurotrophic factors are essential for the survival and differentiation of developing neurons. Recent evidence indicates that neurotrophins, a family of proteins related to nerve growth factor, may also participate in activity-dependent modification of synaptic connections. Using rat hippocampal cultures and Xenopus nerve-muscle cultures as model systems, the applicants propose to investigate the spatial and temporal properties of neurotrophic secretion and action at the synapse. In part I, the applicants will examine acute pre- and postsynaptic effects of exogenous brain-derived neurotrophic factor (BDNF) on the basal synaptic properties and activity-induced plasticity at hippocampal and neuromuscular synapses. Using local delivery of BDNF and BDNF-coated beads, the applicants will determine the spatial and temporal patterns in the BDNF action. The applicants will also examine whether electrical activity affects the action of BDNF by altering the binding, transduction, or internalization of the factor. The potential "gating" and "synergistic" effect of cAMP-dependent processes on the BDNF action will also be explored. In part II, the applicant will examine whether secretion of BDNF from neurons overexpressing BDNF or endogenous secretion of TrkB ligands can modulate synaptic efficacy, whether synaptic activity can trigger a localized secretion of BDNF, and whether the action of secreted BDNF is restricted to the site of BDNF secretion. The trafficking and secretion of BDNF will also be followed by using fluorescently tagged BDNF. Finally, in part III, they will examine the long-term actions of BDNF on the structure and function of the synapse, determine whether such long-term actions can retain synapse-specificity, whether active transport, Ca2+ signaling, gene activation and protein synthesis, and activation of protein kinases and phosphotases are involved in the transduction of long-term BDNF effects. Finally, the applicants will examine the possibility that selective synaptic "tag" associated with long-term specific synaptic modification is due to an activity-dependent upregulation of the receptiveness of the synapse to the action of neurotrophic. Taken together, these in vitro studies provide unique opportunities to address several fundamental questions concerning the role of neurotrophic in synaptic plasticity, and promise to contribute new information relevant to our basic understanding of the nervous system.

DESCRIPTION (Investigator's abstract): The development and plasticity of the nervous system involve activity-dependent modification of synaptic connections. Using simple networks of hippocampal neurons in culture, the applicant recently found that long-term depression (LTD) induced by repetitive synaptic activity at one synaptic site is accompanied by an extensive but selective propagation of the depression to other synapses within the network. The P.I. now proposes to characterize further this phenomenon of propagation of synaptic modification and to examine underlying cellular and molecular mechanisms. The experiments in Aim 1 will determine the conditions by which LTD and long-term potentiation (LTP) can be reliably induced at glutamatergic and GABAergic synapses in hippocampal cultures and the mechanisms underlying the induction and expression of these modifications. The experiments in Aim 2 will further confirm the earlier findings on the back- and lateral propagation of synaptic depression following the induction of LTD and extend these studies to include the propagation of synaptic modification accompanying the induction of LTP. The applicant will determine the time course, extent and persistence of propagated changes, their dependence on the distance from the site of LTP/LTP induction, and pre-and/or postsynaptic mechanisms underlying the changes at the propagated site. In Aim 3, the applicant will examine whether a synapse can achieve temporal integration of multiple modulatory signals propagated from another synapse associated with the same neuron which is undergoing sequential LTD/LTP and spatial integration of multiple signals propagated from different synapses undergoing separate LTD/LTP. Finally, in Aim 4 , the applicant will study the involvement of various forms of extra-and intracellular signaling mechanisms in the propagation of synaptic modification following the induction of LTD/LTP. Together, these studies address several fundamental issues concerning the distribution of activity-induced modifications within a neural network. Multiple whole-cell recording from defined neural networks pioneered in this project promises to uncover previously unknown network properties relevant to our basic understanding of the nervous system.

9808723 POO During the early development of the nervous system, the sensory inputs from the environment received by the central nervous system play a critical role. Abnormal sensory inputs result in abnormal development of nerve connections in the brain. However, how sensory inputs affect these connections is poorly understood. In this project, Dr. Poo and a graduate student and postdoctoral fellow will use an electrical recording method that can detect the efficiency of nerve connections in the intact visual system of the developing frog tadpole. In particular, they will examine how nerve connections from the retina to its major target in the brain, the tectum, are affected by electrical activity in the visual inputs. In Part I of the project, they will determine how repetitive activity induced by electrical stimulation of retinal neurons affects the efficiency of the synaptic connections between the retina and tectum, or retino-tectal synapses. The patterns of electrical activity and the cellular mechanisms that produce long-term changes in efficiency, namely long-term potentiation and/or depression (LTP & LTD) will be examined. In Part II, they will use light stimuli to initiate localized retinal activity and examine whether physiological visual stimuli can induce LTP or LTD of retino- tectal synapses, and if so, how these light-induced synaptic modifications relate to those produced by direct electrical stimulation. In Part III, they will carry out simultaneous electrophysiological and optical recording of identified retino- tectal projections in the tectum, using a newly developed high- resolution microscopic technique called two-photon laser scanning fluorescence microscopy. They will determine whether and how electrical and visual stimuli shape the pattern of axonal processes of retinal ganglion neurons and dendritic processes of the postsynaptic tectal cells. This project offers unique opportunities for direct examination of the effects of se nsory activity on the structure and function of synaptic connections in an intact visual system. The information obtained will be valuable to our understanding of the development of vertebrate visual systems and plasticity of neuronal connections in general.

DESCRIPTION (provided by applicant): Diffusible guidance molecules are responsible for guiding growing axons towards their target cells in the developing nervous system. Several families of guidance factors and their receptors have been identified and the signaling mechanisms are beginning to be elucidated. In this project, we propose to continue our in vitro study of signal transduction events underlying the turning responses of the axonal growth cones of cultured Xenopus spinal neurons and rat cerebellar granule cells induced by microscopic gradients of two guidance molecules, netrin-1 and brain-derived neurotrophic factor (BDNF), and by a secreted form of myelin-associated glycoprotein (MAG). In part I, we will continue our study of the spatiotemporal profiles of Ca2+ and cAMP signals at the growth cone triggered by guidance cues, and the causal relationship betweenCa2+ and cAMP signals that lead to attractive and repulsive turning of the growth cone. In Part II, we will examine how Ca2+ elevation triggered by the activation of the membrane receptors of guidance factors is linked to the activation of the Rho family of small guanosine triphosphatases (GTPases), which are known to regulate the cytoskeletal rearrangements underlying growth cone turning. In Part III, we will examine the initial cytoplasmic events triggered by a gradient of guidance factors that are likely to serve for the amplification of guidance signals, with particular focus on phosphoinositol 3 kinase and its effector molecules that may undergo rapid accumulation towards the leading edge of the growth cone. The role of Ca2+ signaling and Rho GTPases in the amplification process will also be addressed. In Part IV, we will further examine the phenomenon of adaptation (desensitization and resensitization) of growth cone responses in the presence of a gradient of the guidance factor, and the cellular mechanisms underlying the desensitization and resensitization processes. In particular, we will examine how local translation of specific proteins at the growth cone participates in the adaptation process. Together, these studies offer a unique opportunity for elucidating the cellular transduction events at the growth cone underlying the action of extracellular diffusible guidance molecules. The results will contribute to our understanding of the development of the nervous system and provide insights into potential therapeutic approaches for promoting nerve regeneration after injury.

DESCRIPTION (provided by applicant): Neurotrophins (NTs), a family of proteins essential for the survival and differentiation of developing neurons, have been shown to participate in activity-dependent synaptic plasticity. In this project, we propose to test the hypothesis that secretion of NTs at developing synapses is triggered by synaptic activity in a spike pattern-dependent manner and localized synaptic actions of secreted NTs in turn produce long-term functional and structural modification of developing synaptic connections. We will focus our attention on brain-derived neurotrophic factor (BDNF), a NT widely expressed in the developing nervous system, using a combination of physiological, molecular biological, and optical imaging methods. The proposed research consists of three parts. In PART I, we will use cultures of rat or mouse hippocampal neurons to examine the acute pre- and postsynaptic effects of exogenously-applied BDNF on the function and plasticity of glutamatergic and GABAergic synapses and on axon/dendrite morphology and synaptogenesis. The synapse-specific local actions of BDNF and underlying signaling mechanisms will also be investigated. In PART II, we will study the role of endogenously-secreted BDNF in activity-induced modifications of synaptic function and morphology in hippocampal cultures, activity-dependent BDNF secretion, and intra- and interneuronal trafficking of BDNF. In PART Ill, using developing Xenopus tadpoles, we will examine the in vivo effect of BDNF on the function and plasticity of retinotectal synapses, axon/dendrite dynamics and synaptogenesis in the tectum, and the trafficking and secretion of endogenous BDNF. Finally, we will investigate the role of BDNF in the development of retinotopic map and direction-selective receptive field of tectal neurons. Taken together, these in vitro and in vivo studies provide unique opportunities to address several fundamental cell biological issues concerning the modulatory function of neurotrophins in activity-dependent refinement of developing neural connections, and are likely to yield new information relevant to our basic understanding of physiology and pathology of the developing nervous system.

DESCRIPTION (provided by applicant): In the developing visual system, the spatiotemporal patterns of visual inputs play an important role in shaping the connections of developing circuits. Using the developing retinotectal system of Xenopus tadpoles and a combination of in vivo whole cell recording and optical imaging techniques, we proposed to examine the development and experience-dependent refinement of visual circuits at the cellular level. In particular, we will examine the potential link between activity-dependent persistent synaptic modifications known as long-term potentiation/depression (LTP/LTD) and developmental refinement of visual circuits. The proposal consists of four parts. In PART I, we will examine the time course of synaptogenesis in the tectum, developmental changes in the synaptic function and plasticity and in the receptive field properties of tectal neurons, using a whole-cell recording method that allows the selective assessment of changes in glutamatergic and GABAergic/glycinergic inputs to the tectal neuron. The modulatory influence of GABAergic/glycinergic inputs on the development of receptive field properties will also be studied. In PART II, we will examine the effects of visual experience-evoked activity on retinotectal synapses and on the development of retinotopic map and receptive field properties. We will also determine the role of spike-timing dependent plasticity in the activity-induced refinement of retinotectal connections. In PART III, we will examine the relationship between activity-induced LTP/LTD and the structural refinement at identified retinotectal connections and the role of cooperative and competitive interactions among converging retinal inputs on the dendrite of single tectal neurons. Finally, in PART IV, we will examine the process by which visual input-induced LTP/LTD and structural modifications of retinotectal connections can be consolidated and become resistant to disruption by subsequent spontaneous spiking activity in the retinotectal system. Taken together, these in vivo studies provide unique opportunities to address several fundamental issues concerning the instructive function of sensory inputs in the refinement of developing neural connections, and are likely to yield new information relevant to our basic understanding of physiology and pathology of the developing nervous system.

[unreadable] DESCRIPTION (provided by applicant): The development and function of the nervous system depend on activity-dependent modification of synaptic connections. Correlated spiking of pre- and postsynaptic neurons can result in persistent strengthening or weakening of synapses, termed long-term potentiation (LTP) or long-term depression (LTD), respectively, depending on the temporal order of pre- and postsynaptic spiking. Our recent results showed that correlated spiking that induces LTP or LTD also leads to bi-directional changes in the intrinsic excitability of the presynaptic neuron and in spatial summation of excitatory postsynaptic potentials (EPSPs) in the postsynaptic dendrite. Furthermore, LTP or LTD induced at one synaptic connection was found to spread to other synaptic sites within the circuit. In the present project, we propose to continue our research on spike timing-dependent plasticity (STDP) of synapses and neuronal properties, using dissociated hippocampal cell cultures and acute hippocampal and cortical slices. In Part I, we will further examine the properties of STDP of glutamatergic and GABAergic synapses in hippocampal circuits, the cellular mechanisms underlying the expression of LTP/LTD, and the factors that modulate the time windows of pre- and postsynaptic spiking for the LTP/LTD induction. In Part II, we will examine the effects of correlated on the intrinsic excitability of both pre- and postsynaptic neurons in cortical slices and on spatial and temporal summation of EPSPs in the dendrites of CA1 pyramidal cells in hippocampal slices. In Part III, we will examine the non-local nature of synaptic modifications following LTP/LTD induction by correlated activity, i.e., the heterosynaptic effects and the spread of potentiation/depression within the neural circuits in hippocampal cultures or in acute hippocampal and cortical slices. Together, these studies address several fundamental issues concerning STDP of neural circuits and explore new territories of neuronal plasticity. The information and insights obtained will contribute to our understanding of the development and plasticity of the nervous system. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): These experiments will chart the connections between the inferior colliculus (IC), medial geniculate body (MGB) and auditory cortex (AC), three structures essential for normal hearing. This will enable us to propose hypotheses about the nature of the tectothalamocortical transformation. Three studies are proposed. First, we will analyze connections within the IC that permit its several subdivisions to communicate, with emphasis on structures other than the central nucleus. We will then document the types of IC neurons containing gamma-aminobutyric acid in immunocytochemical analyses. Second, we will trace IC connections to the MGB using anterograde and retrograde tracers. This aim contrasts and compares the projection patterns of IC subdivisions with their MGB targets, revealing more fully the thalamic projections of midbrain souces. Finally, we will label and identify the AC cells that descend to the IC and MGB since these pathways may be important in processing biologically salient sounds. These experiments use anesthetized rats and cats to document how these structures interact. The cat is also used in collaborative physiological-anatomical studies. The ultimate goal of this work is to create plausible, principled, and data-based biological models of auditory midbrain, thalamic, and cortical connectivity that incorporate specific projections and local circuits in each of their subdivisions. There is reason to believe that the several subdivisions of the IC each have a unique role to play in hearing, in multimodal integration, and in auditory relations to the limbic system. Placing these roles in a functional hierarchy can clarify the relations between hearing and behaviors such as startle reflexes and audiogenic seizures, processes which likely depend on interactions between auditory midbrain, thalamus, and cortex for their expression. Such models precede more refined theories of auditory system relations with the limbic and motor systems, to which hearing is related closely by connections and function. Damage from stroke, aging or traumatic brain injury can cause deficits in hearing and in speech comprehension. Animal models are an essential step towards understanding effective clinical interventions. At present only hearing aids and cochlear prostheses can be used to ameliorate deafness. It may ultimately be possible to craft central prostheses that could be implanted in IC, MGB, and AC to stimulate the brain directly in attacking the problem of hearing loss. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The long-term goal of our research is to understand the neural circuit basis of learning and memory. In this project, we aim to understand how neural circuits process and store temporal sequence information in the sensory experience. We propose to test the specific hypothesis that spike timing-dependent plasticity (STDP) of intracortical connections provides a mechanism for cortical circuits to perform sequence learning. The hypothesis is based on our preliminary finding that repetitive visual conditioning with uni-directional moving spot in the visual field of either anesthetized or awaked rats resulted in sequential spiking of a specific ensemble of V1 neurons with adjacent receptive fields along the path of the moving spot, and this neuronal ensemble retained the memory of sequential spiking, as manifested by post-conditioning evoked and spontaneous sequential spiking of the ensemble. To test our hypothesis, we propose to carry out experiments on head-fixed awake and anesthetized rats, using a combination of in vivo techniques that include multielectrode array recording, voltage-sensitive dye imaging (VSDI), in vivo whole-cell recording, and optogenetic neuronal activation. In Aim 1, we will confirm and characterize the phenomenon of sequence learning and recall/replay and determine the optimal parameters of visual conditioning in inducing sequence learning/memory, as monitored by the multielectrode array and VDSI of cortical activity waves. In Aim 2, we will determine whether conditioning visual stimuli can indeed induce synaptic modification of intracortical excitatory connections in a manner consistent with STDP, and whether the induction and expression mechanisms of visual conditioning-induced synaptic changes are similar to STDP induced by repetitive pre- and postsynaptic neuronal spiking. In Aim 3, we will determine whether STDP is necessary for visual conditioning-induced sequence learning and sufficient by itself for inducing sequence memory in the V1. Together, this project will provide new insights into the synaptic and circuit mechanisms by which temporal sequence information may be coded and stored by cortical circuits. It will also advance our understanding of the properties of STDP in vivo and its role in learning and memory functions. PUBLIC HEALTH RELEVANCE: The temporal sequence of events is a critical element in learning and memory. This project will provide new insights into the synaptic and circuit mechanisms in the cortex underlying sequence learning and memory.