Donald Arnold - US grants

DESCRIPTION (provided by applicant): The purpose of the studies proposed in this grant is to understand the molecular mechanisms underlying polarized targeting of neuronal transmembrane proteins. The specific aims of this grant will concentrate on three main levels of polarized targeting: 1. Targeting of proteins at the plasma membrane through compartment-specific endocytosis and vesicle docking. 2. Trafficking of vesicles containing either axonal or dendritic proteins to the appropriate polarized compartment. 3. Sorting of axonal and dendritic proteins into separate vesicles. The ultimate goal of this research is to understand how these targeting mechanisms produce surface distributions of proteins that are highly polarized. Relevance Protein targeting plays an important role in many physiological processes such as the establishment and maintenance of synaptic connections. In addition, defects in trafficking are associated with numerous neurological diseases of major clinical significance such as Alzheimer's and Huntington's disease.

DESCRIPTION (provided by applicant): Studying the localization of proteins with conventional antibodies has greatly contributed to our understanding of the structure and function of neurons. However, conventional antibodies have several limitations that drastically limit their utility. Tissue must be fixed and permeabilized prior to staining and often the overlapping expression patterns of adjacent neurons are difficult to interpret because of the lack of contextual information. For these reasons, the precise subcellular localization patterns in vivo of the majority of neuronal proteins have not been well characterized. The purpose of the studies proposed in this grant is to develop genetically encoded probes that will allow the subcellular localization of neuronal proteins to be mapped in vivo and in real time with high fidelity. These probes consist of genetically encoded aptamers (intrabodies) that bind to endogenous neuronal proteins and are generated using the mRNA display system. Three different types of intrabodies will be generated: 1. Binders to individual cytoskeletal proteins that mark neuronal structures such as pre- and postsynaptic sites. 2. Binders to transmembrane proteins. These intrabodies will be modified to enable them to label either total protein or only protein that is present on the plasma membrane of the cell. 3. Binders to activated G-proteins. Intrabodies will be used to attach three types of molecules to endogenous target proteins: 1. Fluorescent molecules that can be used to report the localization of the protein. 2. proteins for measuring Ca++ concentration in the region around the protein. 3. proteins that are activated by light to produce depolarizing currents. Subcellular trafficking of proteins is crucial to virtually all neuronal functions, including establishment of synaptic connections, axon guidance and synaptic plasticity. Disruption of protein trafficking has been linked to such diseases as Alzheimer's disease and Parkinson's disease. Protein trafficking also plays a critical role in drug addiction. Intrabodies generated through RNA display will provide tools to map the subcellular localization of endogenous proteins with high fidelity, in vivo and in real time, which is not possible with current technology.

DESCRIPTION (provided by applicant): The goal of this proposal is to develop methods to target light-activated proteins, such as Channel Rhodopsin II (ChR2) and Halorhodopsin (NpHR), to specific subcellular compartments in neurons. Both ChR2 and NpHR generate electrical currents in response to light: ChR2, an ion channel, passes depolarizing cation current in response to blue light, whereas NpHR, a pump, generates hyperpolarizing Cl- currents in response to yellow light. Neurons expressing these proteins can be efficiently excited or inhibited with light. ChR2 and NpHR can thus be expressed in specific neuronal populations to determine if activity in these cells is sufficient and necessary to drive a function, for example a behavior. ChR2 has been used to map synaptic circuits in brain slices, by combining patch clamp recording of postsynaptic cells with stimulation of presynaptic neurons expressing ChR2. ChR2 can also be used to stimulate neurons in a spatial pattern in vivo, for example to determine motor maps. A major limitation to the use of light-activated proteins in circuit mapping is their tendency to localize nonspecifically to different neuronal compartments. ChR2 and NpHR appear to be expressed equally well in axons and dendrites. In most neural tissues, dendrites and local and long-range axons are intermingled. The presence of these proteins in axons means that photostimulation can have non-local effects. Thus, it is virtually impossible to stimulate dendrites from one ChR2-positive cell without also stimulating neighboring ChR2-positive axons that can arise from distant and functionally unrelated neurons. Similarly, action potential propagation can be blocked by photostimulation of NpHR-positive axons. It is therefore of great interest to generate versions of NpHR and ChR2 that can be excluded from axons, by targeting them to dendrites and somata. In other applications it is advantageous to specifically target light- activated proteins to axons. Here we propose to generate peptides encoding signals that target light-activated proteins to specific subcellular compartments allowing neurons to be activated or inhibited for neural circuit analysis.

DESCRIPTION (provided by applicant): The purpose of the research proposed in this grant is to generate tools that will allow endogenous synaptic proteins to be visualized in neurons in vivo. Previously we have generated recombinant antibodies known as FingRs (Fibronectin intrabodies generated with mRNA display) that can be expressed in living neurons where they label endogenous target proteins noninvasively and with high fidelity. Furthermore, a transcriptional regulation system controls the expression levels of FingRs so that they are expressed at precisely the same level as their endogenous counterparts, insuring low background. In this grant we will generate transgenic mice that express fluorescent protein-fused FingRs that recognize synaptic proteins. Synaptic proteins in individual neurons in the brains of these mice can be visualized in vivo in real time. In turn, this will allow events at the molecular level to be correlated with events at the cellular, circuit and whole animal level. In particular, by mapping the locations and amounts of synaptic proteins in neurons, it will be possible to monitor the strength of both synaptic inputs and outputs in living neurons. The ability to monitor synaptic strength will be very useful for studying diseases associated with aberrant synaptic connectivity including schizophrenia, mental retardation and autism.

DESCRIPTION (provided by applicant): Here we propose to develop an experimental paradigm to allow dynamic monitoring of the strength and location of every glutamatergic and GABA/Glycinergic synapse within the brain of a living organism. In combination with behavioral manipulation of the organism this paradigm will allow for study of how the brain encodes information in synaptic structure. This paradigm will involve combining three technologies: 1. Recombinant probes with which postsynaptic excitatory and inhibitory sites can be labeled in vivo, allowing the location and strength of synaptic connections to be monitored in parallel. 2. 2P-SPIM microscopy, which can image large volumes very quickly, without bleaching and, potentially, with isotropic resolution. 3. Software to calculate and store the location and strengt of each synapse in such a manner that it can be easily manipulated and analyzed. Experiments will be performed in zebrafish, as they have semi-transparent brains that are relatively small, yet they are capable of relatively complex behaviors. Experiments will be used both to establish the viability of the paradigm and to answer fundamental questions about how synapses are modulated during sleep, as well as how they are changed in learning paradigms such as sound habituation and place preference/aversion conditioning.

DESCRIPTION (provided by applicant): The purpose of the experiments proposed in this grant is to generate recombinant antibody-like proteins (intrabodies) that can label specific proteins of all types, including cytoplasmic and nuclear proteins, and extracellular epitopes on transmembrane proteins. Recently we have shown the utility of the intrabody approach by generating probes that recognize the postsynaptic proteins Gephyrin and PSD95. These probes work efficiently and can be used to visualize endogenous proteins in living organisms. However, the design of these intrabodies places constraints on the targets against which they can be made. In particular, target proteins must be fixed to the cytoskeleton of the cell. Here we propose to develop a new strategy for generating intrabodies that does not place any requirements on the type of target protein. To establish the efficacy of this strategy we will generate intrabodies against the motor protein Kinesin 1, the transcription factor AP-1 and an extracellular epitope of the AMPA receptor GluA1.

DESCRIPTION (provided by applicant): Here we propose to develop an experimental paradigm to allow dynamic monitoring of the strength and location of every glutamatergic and GABA/Glycinergic synapse within the brain of a living organism. In combination with behavioral manipulation of the organism this paradigm will allow for study of how the brain encodes information in synaptic structure. This paradigm will involve combining three technologies: 1. Recombinant probes with which postsynaptic excitatory and inhibitory sites can be labeled in vivo, allowing the location and strength of synaptic connections to be monitored in parallel. 2. 2P-SPIM microscopy, which can image large volumes very quickly, without bleaching and, potentially, with isotropic resolution. 3. Software to calculate and store the location and strengt of each synapse in such a manner that it can be easily manipulated and analyzed. Experiments will be performed in zebrafish, as they have semi-transparent brains that are relatively small, yet they are capable of relatively complex behaviors. Experiments will be used both to establish the viability of the paradigm and to answer fundamental questions about how synapses are modulated during sleep, as well as how they are changed in learning paradigms such as sound habituation and place preference/aversion conditioning.

DESCRIPTION (provided by applicant): The purpose of the experiments proposed in this grant is to generate recombinant antibody-like proteins (intrabodies) that can label specific proteins of all types, including cytoplasmic and nuclear proteins, and extracellular epitopes on transmembrane proteins. Recently we have shown the utility of the intrabody approach by generating probes that recognize the postsynaptic proteins Gephyrin and PSD95. These probes work efficiently and can be used to visualize endogenous proteins in living organisms. However, the design of these intrabodies places constraints on the targets against which they can be made. In particular, target proteins must be fixed to the cytoskeleton of the cell. Here we propose to develop a new strategy for generating intrabodies that does not place any requirements on the type of target protein. To establish the efficacy of this strategy we will generate intrabodies against the motor protein Kinesin 1, the transcription factor AP-1 and an extracellular epitope of the AMPA receptor GluA1.

Although considerable information about neuronal circuits has been generated from experiments where retrograde transsynaptic tracing has been performed using Rabies Virus, there is no comparable technique for transsynaptic tracing in the anterograde direction. The purpose of this grant is to optimize and validate a method for mediating anterograde transsynaptic tracing from genetically or physiologically determined cells. This method is monosynaptic, only marks cells that are postsynaptic to the starter cells and has negligible toxicity. To validate this method we will use a combination of immunocytochemistry, optogenetics and electrophysiology, and we will perform experiments both in vitro and in vivo. In addition, we will develop methods for tracing both excitatory and inhibitory transsynaptic connections. This method has the following advantages over current methods of transsynaptic tracing: 1. All steps of the process are well-defined and thus can be independently tested and optimized. 2. It is mediated by a single receptor, so that when applied to neurons such as dopaminergic neurons that release more than one neurotransmitter, it should be possible to independently trace pathways mediated by each transmitter. 3. It should be generalizable to any postsynaptic receptor, allowing many different pathways to be explored. 4. The receptor that mediates transsynaptic labeling is physiologically relevant ? only electrically active synapses, and not silent synapses, will mediate postsynaptic labeling.

Classical conditioning has been studied in many different animal models, and even in humans. However, the larval zebrafish with its transparent brain offers a unique opportunity to observe large scale changes in synaptic structure that accompany this form of learning. Accordingly, we have developed a novel paradigm for visualizing synaptic changes that occur during classical conditioning in larval zebrafish. Using this paradigm we have observed striking region-specific changes in the distributions of synapses that drive the rewiring of neural circuits that mediate threat responses. In this grant we will expand this paradigm by monitoring neuronal activity through imaging of genetically encoded calcium indicators throughout the pallium (the homolog of the amygdala) before, during and after classical conditioning and extinction. This will allow us to identify cells that comprise the circuits that control threat and safety and explore their connectivity using optogenetics. We will investigate how different sensory inputs can cause changes in the activity of those cells leading to synapse change, and the formation or extinction of associative memories. A crucial component of these studies will be the recording of field potentials to capture rhythmic activity throughout the pallium and high speed SPIM imaging of genetically encoded voltage indicators to record rhythms in individual cells. By understanding the precise timing of signals that impinge on individual cells we will uncover mechanisms that underlie synaptic plasticity. Our goal is to develop a theoretical model describing the neural circuits that underlie threat detection and how they can change as a result of associative memory formation and extinction.