Gina G. Turrigiano - US grants

It is generally accepted that the abilities to learn and remember arise through activity-dependent modifications in the properties of the neural circuits underlying behavior. The outputs of neural circuits depend on an interplay between synaptic connections and the intrinsic firing properties of individual neurons, but despite the importance of these intrinsic neuronal properties for circuit function, studies on the mechanisms of learning have stressed almost exclusively the role of synaptic modifications. We have shown that isolated lobster stomatogastric ganglion neurons in culture undergo activity-dependent transitions between tonic firing and burst firing states in a calcium-dependent manner. These transitions are produced by regulation of the expression of ionic conductances. These studies indicate that the intrinsic firing properties of neurons, like synaptic strengths, can be continuously modified by ongoing activity, and that the firing properties of a neuron are in part a function of its recent history of activation. This proposal will follow up on these findings by using electrophysiological and calcium imaging techniques to address the mechanisms by which activity modifies intrinsic neuronal firing properties. The role of intracellular calcium, protein synthesis, and protein kinases in this process will be addressed. We will determine whether local activation of individual neurites can locally modify the magnitude or distribution of conductances, and whether this modification acts to oppose or to potentiate synaptic inputs. The long-term goal of this proposal is to understand how intrinsic neuronal properties and synaptic strengths are conjointly regulated to encode experience. In order to study the interactions between intrinsic and synaptic plasticity, we have begun to develop a cortical culture system in which the firing properties and synaptic properties of specific populations of neurons can be studied, and where spontaneous synaptic activity is high. The additional time for research afforded me by the award of a K02 grant will allow me concentrate my personal effort on developing this new culture system. I believe this system will provide essential information about the building blocks of learning and memory, and the role of experience in determining and modifying nervous system structure and function.

9726944 TURRIGIANO Much of the development of the mammalian central nervous system occurs after birth, and requires experience in order for functional circuits to form. A central problem in neurobiology has been to elucidate the rules by which experience, in the form of patterned neural activity, determines which synaptic connections are maintained and which are lost. An important hypothesis is that neurons compete with each other in an activity-dependent manner for the establishment of synaptic connections. This is thought to occur through competition between synapses for limiting quantities of a neurotrophic agent that is essential for maintenance of the synapse. Presynaptic neurons that are successful at exciting the postsynaptic neuron obtain more neurotrophic agent and therefore out-compete neurons that are not. Although this model has generated much interest, it has not yet been directly tested. Cortical circuits have both inhibitory and excitatory synaptic connections. Inhibitory connections, where electrical activity in a presynaptic neuron inhibits electrical activity in the postsynaptic neuron, is essential for the proper functioning of cortical circuits, but despite this importance most research to date has concentrated almost exclusively on elucidating the rules by which excitatory synaptic connections are formed. While the rules for formation of inhibitory connections have not been established, an understanding of these rules is crucial for understanding the genesis of cortical circuits. The goal of Dr. Turrigiano's research project is to ask whether the formation of inhibitory synaptic connections involves competitive mechanisms, and to ask whether this competition occurs through the activity-dependent uptake of a trophic agent. These experiments will help to complete our understanding of neural circuit development, and will test a prominent model for how activity wires up the mammalian central nervous system.

DESCRIPTION (provided by applicant): We request funds for the purchase of a Leica Spectral Confocai-Multiphoton Microscope (Leica SP2 MP) for combined imaging and electrophysiological experimentation. There is no multiphoton microscope at Brandeis, and our single confocal microscope cannot be used for combining imaging and electrophysiology. Such an instrument would greatly facilitate the N.I.H. funded research of the user group. It will also benefit other life science researchers at Brandeis interested in live imaging of biological material. The research of the five investigators in the user group is now well poised to take advantage of combined confocal/multiphoton imaging and electrophysiology. Projects include: mechanisms of synapse formation, synaptic plasticity, neuromodulation, and the regulation of intrinsic excitability. Four of the five investigators have a track record in confocal and quantitative imaging, and a different subset of the four have extensive electrophysiological experience. All five investigators have reached the limit of what can be achieved without an integrated system for combining electrophysiology and live imaging. The Leica SP2 MP instrument would be housed in the Imaging Facility that currently houses our confocal. Because we have had a confocal facility for 10 years, mechanisms of operation and maintenance are already in place. The Imaging facility is managed by microscopist with 20 years of experience. He will commit 50% of his time to running and maintaining the SP2 MP, and will be responsible for training as well. Brandeis University and the Biology Department are committed to maintaining this facility. In addition to the user group, the confocal/multiphoton facility will be available to all life science researchers at Brandeis who have a need for this technology on a time-available basis.

DESCRIPTION: Sensory experience plays an important role in refining the connectivity of primary visual cortex, but the identity of the synaptic plasticity mechanisms that contribute to this refinement are still under debate. Most research has concentrated on the role of correlation-based plasticity mechanisms such as LTP and LTD, but these mechanisms are highly destabilizing and are unlikely to be sufficient to explain all of activity-dependent development. Using cultured cortical networks we identified a novel form of homeostatic synaptic plasticity that scales excitatory and inhibitory synaptic strengths up and down in the correct direction to stabilize the activity of cortical networks, and more recently we have demonstrated a similar phenomenon in vivo. Here we propose to examine the role of this homeostatic synaptic scaling in experience-dependent plasticity in vivo using a classic sensory deprivation paradigm, monocular deprivation (MD) and binocular deprivation (BD) using lid suture. MD and BD have been used extensively to study activity-dependent plasticity, but the effects of these manipulations on intracortical circuitry have never been probed in detail. We will approach this problem by manipulating activity in rodent visual cortex through MD and DR, then cutting slices of primary visual cortex and obtaining whole-cell recordings to measure quantal currents and paired synaptic transmission. We will ask whether the quantal currents for excitatory and inhibitory synapses are scaled in the opposite direction in response to altered visual input, whether this scaling displays critical periods as do other forms of activity-dependent plasticity, and whether the rules for synaptic scaling are specific for particular classes of excitatory and inhibitory inputs. These experiments will lay an important foundation for understanding the detailed changes in cortical circuitry that arise as a result of altered sensory experience, and will have important implications for the mechanisms of visual abnormalities such as amblyopia.

DESCRIPTION (provided by applicant): The Gordon Research Conference (GRC) on Neural Circuits and Plasticity (formerly Neural Plasticity) has been held in alternate years since 1977. We are requesting partial support for the June 26-July 1, 2005 meeting, and the 2007 and 2009 meetings, at Salve Regina University in Newport, Rl. The GRCs were established to stimulate scientific interchange in an informal setting. Interchange is promoted by the informal nature of the conference and by the large amount of time committed to questions during the sessions. The GRC rule prohibiting publication or citation of the meetings and presentations promotes open discussion of the latest results and current ideas. This format has been particularly useful for the Conference on Neural Circuits and Plasticity, a highly interdisciplinary meeting in which the formation, function, and plasticity of neural circuits is explored at the molecular, cellular, systems, and computational levels. The 2005 Conference will have 9 scientific sessions, with a keynote talk by Dr. Bert Sakmann, a Nobel Laureate and one of the most renowned scientists studying circuit function and plasticity. The scientific sessions will cover a range of topics of current interest in the field, from synaptogenesis to sensory coding. The speakers and session chairs are all either world leaders in their fields or up-and-coming junior investigators. Afternoon poster sessions provide an opportunity for all interested participants to present and discuss their work, and each session will include 1 or 2 short talks selected from the submitted abstracts. This format worked very well in previous years to promote young investigators. Question and answer periods are generously scheduled, and social events will permit new participants to meet speakers and session chairs informally. Past participants have found these informal interactions to be a highlight of the meeting, and an important source of scientific advancement for junior people. We are requesting support to fund the attendance of underrepresented groups in the field of Neural Circuit plasticity, including women and ethnic minorities.

DESCRIPTION (provided by applicant): The proper functioning of the mature visual system depends upon experience-dependent refinement of cortical circuitry during early postnatal development. Visual deprivation during this early critical period (CP) can lead to loss of visual responsiveness to the deprived eye (amblyopia). Despite decades of research it is still unclear which cellular plasticity mechanism(s) contribute to this loss of visual responsiveness, and by extension are required for the normal development of visual function. For example, long-term depression (LTD) of cortical excitatory synapses has been suggested to be necessary and sufficient for loss of visual responsiveness following MD, while other studies have raised the possibility that potentiation of inhibition plays a critical role in this process. Recently the maturation of inhibition from fast-spiking GABAergic basket cells (FS cells) has been implicated in the regulation of CP onset, but why mature inhibition is necessary for CP plasticity is still unknown. During the past 2 funding periods we found that monocular deprivation (MD) during the rodent CP dramatically potentiates inhibitory transmission at FS synapses onto star pyramidal neurons (SP, the major excitatory cell type within L4 of rodent V1), by inducing a novel form of long-term potentiation of inhibition (LTPi) at this synapse. Further, LTPi is developmentally regulated, turns on coincidently with the opening of the CP, and manipulations that delay or advance the CP also delay or advance the ability of FS synapses to express LTPi. In this proposal we wish to test the central hypothesis that LTPi plays a critical role in the lossof visual responsiveness induced by visual deprivation, so that FS cells initiate the CP by gaining the ability to express LTPi. To this end we will exploit our mechanistic understanding of LTPi to block its induction in vivo, and determine the role LTPi plays in the cortical response to monocular deprivation (MD). Further, we will evaluate the relative importance of LTD and LTPi in MD-induced loss of visual responsiveness within L4, and ask whether these two forms of plasticity are induced in a synergistic manner during MD. We will combine in vivo and in vitro electrophysiology, viral-mediated gene transfer, and optogenetics to achieve these goals. These experiments will settle a long-standing debate about the mechanisms underlying amblyopia, and have the potential to radically alter our view of the cellular underpinnings of CP plasticity. )

My lab pioneered the study of homeostatic plasticity in neocortical neurons and networks. Recently we found that neocortical neurons both in vitro and in vivo have a firing rate set point (FRSP) around which they regulate their average firing. This is accomplished through a set of homeostatic plasticity mechanisms, including synaptic scaling and homeostatic regulation of intrinsic excitability, that act to restore excitability when it is perturbed by changes in sensory drive or synaptic input. We have made significant inroads into understanding the molecular signaling pathways and machinery that accomplishes this homeostatic regulation of firing; in particular, we know that cell-autonomous changes in average neuronal firing that modulate somatic calcium influx lead to changes in signaling through the calcium-dependent kinase CaMKIV, and this in turn produces transcription-dependent homeostatic alterations in the surface expression of ion channels and glutamate receptors. Despite this progress, it remains largely mysterious how neocortical neurons (or any other cell type) build a stable firing-rate set-point out of calcium-dependent signaling pathways. Here we wish to test the central hypotiiesis that FRSP in neocortical neurons is the equilibrium point of opposing calcium-dependent signaling pathways that regulate excitability, and that this basic principle for how to build a stable FRSP wdll generalize to fundamentally different cell types. We have three major aims: i) test the prediction that FRSP can be modulated by altering the strength of specific calcium-dependent signaling pathways, such as CaMKIV; 2) Identify other elements that act in opposition to CaMKIV and together comprise the FRSP, and 3) determine which homeostatic effectors are downstream of CaMKIV to generate homeostatic compensation. This project wdll illuminate the mechanistic underpinnings of a fundamental aspect of neuronal physiology, and through collaborative work the Other Pis on this Program Project.

? DESCRIPTION (provided by applicant): Homeostatic synaptic scaling is an important form of plasticity thought to be essential for maintaining stable function in developing neural circuits. Synaptic scaling scales the strength of all of a neuron's excitatory synaptic strengths up or down in the correct direction to stabilize neuronal firing rates. These homeostatic adjustments in synaptic weights are accomplished in large part through changes in the synaptic accumulation of GluA2-containing AMPAR at synapses, and appear to operate on all excitatory synaptic inputs onto a given neuron in response to changes in the neuron's own firing. Despite great recent interest, the molecular and biophysical mechanisms that enable this homeostatic adjustment of AMPAR abundance during synaptic scaling are still poorly understood, and many of the assumptions underlying this model of synaptic scaling (such as its global nature) remain largely untested. In this proposal we aim to illuminate the mechanisms that lead to enhanced synaptic AMPAR abundance during scaling up, and to test the idea that this form of plasticity acts on all excitatory inputs to stabilize neuronal firing in vivo. This proposal is built around or recent observation that the AMPAR-binding protein GRIP1 is essential for the regulated increase in synaptic AMPAR abundance during scaling up, and that this process requires direct interactions between GRIP1 and GluA2. Here we proposed to determine how (at the biophysical level) this regulated interaction between GluA2-GRIP1 drives an increase in synaptic AMPAR abundance, by using a variety of cutting edge imaging approaches. We will test two alternative models: first, that GRIP1 traffics to synapses along with AMPAR and enhances synaptic capture of the receptor, and second, that GRIP1 enhances synaptic delivery of modified AMPAR that have an enhanced affinity for synaptic scaffolds. Further, we will use the tools we have generated through these in vitro studies to selectively disrupt AMPAR trafficking during synaptic scaling up in vivo, in order to probe the mechanism and function of synaptic scaling within intact neocortical circuits.

PROJECT SUMMARY Primary cilia are sensory organelles that are now known to be present on nearly every neuron type in the mammalian central nervous system. In the developing nervous system, cilia are essential for progenitor proliferation, neuronal migration, and the establishment of synaptic connectivity. However, although cilia are also present on mature neurons, their roles in the postnatal brain are poorly understood. Intriguingly, cilia concentrate neuropeptide and amine receptors, and cilia dysfunction has been linked with multiple neuropsychiatric diseases, suggesting that cilia-dependent neuromodulator signaling may be critical for the maintenance and plasticity of neural circuits. The overall goal of this exploratory R21 is to investigate the role of ciliary signaling in the acute modulation of excitatory synapse formation and function in the postnatal brain. In preliminary experiments, we have found that acute disruption of cilia in postnatal cortical neurons increases excitatory synapse number and strength. Consistent with this finding, spontaneous neuronal firing rates (driven by synaptic input) are also increased indicating a disruption of excitation/inhibition (E/I) balance. Since E/I imbalance contributes to multiple neuropsychiatric and metabolic disorders, our results raise the novel and exciting possibility that ciliary signaling is essential for generating and maintaining correct E/I balance in multiple postnatal circuits. We will take advantage of the complementary expertise of the co-PIs (Sengupta ? cilia biology, Turrigiano ? synaptic physiology) to: Aim 1. Establish a role for ciliary signaling in the regulation of E/I balance and circuit excitability. Aim 2. Explore the mechanisms by which ciliary signaling modulates synaptic properties. Results from this work have the potential to open up new avenues for understanding how correct E/I balance is dynamically maintained in the postnatal brain, and provide insights into how cilia dysfunction contributes to the regulation of mental health.

Project Summary This is a proposal to continue a long-standing postdoctoral training program at Brandeis University. This program integrates intensive quantitative and experimental training in a collaborative environment to produce neuroscientists who go on to successful careers applying these skills to solve the myriad problems posed by disorders of the nervous system. We will take a diverse and talented set of individuals with Ph.D.s in neuroscience, physics, math, computer science, engineering, and biological sciences and give them systematic and integrative training in the use of modern quantitative/analytical approaches bridging levels of analysis to understand nervous system function. We have designed a modular and collaborative training structure that allows trainees with disparate backgrounds to acquire the skills, training and flexibility they need to thrive as modern neuroscientists. Our program comprises a two-year sequence that systematically builds quantitative literacy, fluency with statistical methods and quantitative tools, training in rigor, experimental design and ethics, and grant-writing and communication skills. We intend to recruit 4 Postdoctoral Trainees/year into this 2-year program for a total of 8 slots, which maintains the previous level of support. We will magnify the impact of this training program by encouraging all Neuroscience postdocs, not only those appointed to the training grant, to participate. This will serve the purpose of building a collaborative postdoc community that collectively possesses and disseminates a broad set of quantitative skills, and where horizontal and vertical transmission of these skills will be an important feature of training. The richer this trainee and mentor community is, the more effective the training will be. To this end, we are committed to enhancing the diversity of our training program at all levels, and a set of concrete steps to accomplish this are proposed.