Philip Haydon - US grants

The primary goal of the proposed investigation is the detailed elucidation of the molecular aspects of toxin regulation in V. cholerae. Biochemical and genetic methods will be used to locate the DNA sites involved in formation of the cholera toxin operon (ctx) promoter and control sites. We will attempt to define the precise molecular mechanism by which the positive regulatory gene toxR, activates ctx transcription. The regulatory role played by the repetitive sequence TTTTGAT will be studied by localized and mismatch primer mutagenesis. The nutritional and physical factors which regulate toxin production will be correlated with specific transcriptional effects on ctx or toxR. We will continue to study ctx amplification as a genetic mechanism controlling toxin production in E1 Tor strains of V. cholerae. In this regard, we will test the repetitive sequence RS1 for its ability to mediate various recombinational events including transposition, duplication and site-specific recombination. The knowledge gained from all the above studies will be used to devise methods with which to study toxin regulation and ctx amplification in vivo. Specifically, we will study how the intestine provides a selective environment for increased toxin expression by defining which genes (ctxA, ctxB or toxR) are necessary for in vivo selection of genetic reversion and amplification events. We will apply what we have learned about ctx regulation and expression to the development of improved live and dead, oral vaccines against cholera. Mutations will be constructed in the cloned recA gene of V. cholerae and then recombined back in place of the rec+ gene of the prototype live oral vaccine strain 0395-N1. In addition to recA, streptomycin-dependent, DAP- and ga1E mutations will also be incorporated into 0395-N1 derivatives as a means of limiting the persistence of this strain in humans and the environment. Finally, derivatives of 0395-N1 which are suitable for use as a dead, oral cholera vaccines will be constructed which hyperproduce the B subunit of the toxin in both a secreted and nonsecreted form.

The biochemical and genetic properties of staphylcoccal enterotoxin A (SEA) will be characterized. Knowledge gained from the nucleotide sequence of the SEA structural gene (entA) will be used to perform site-specific mutagenesis of the entA gene in regions likely to encode amino acid residues involved in formation of the SEA active site or receptor binding domain. The mutant entA gene products will be assayed for emetic activity, mitogenic activity and interferon induction. If these studies lead to the identification of regions involved in the formation of SEA's biological active site(s), then short synthetic peptides homologous to these regions will be tested for the induction or inhibition of biological activities. These studies could lead to the development of SEA toxoids, and possibly new pharmacological agents for the induction of interferon or mitogenesis. The true incidence of SEA production in clinical isolates of S. aureus will be determined using an entA gene probe. In addition, the active site studies discussed above may also result in the development of an oligonuleotide probe (directed at homologies present in the three enterotoxins SEA, SEB and SEC) that might identify all enterotoxin producing Staphylococcus aureus strains in a hybridization assay. To determine if SEA can contribute to the pathogenicity of S. aureus strains, the relative virulence of isogenic EntA+ and EntA- S. aureus strains will be tested in several animal models. Characterization of the entA-converting phages and related non-entA converting phages will continue with respect to genetic properties and genome structure. These studies may lead to a better understanding of how entA-converting phages arise in nature and the mechanism by which antigenic heterogeneity is generated in the staphylococcal enterotoxins. The possibility that entA gene expression is regulated will be examined by assessing the effects of strain background, media composition, and gene dosage on a chloramphenicol-entA-promoter fusion.

Synaptogenesis is a developmental process fundamental to the establishment of neuronal networks. However, there is relatively little knowledge of the electrophysiologically detectable events occurring during synaptogenesis, and little knowledge concerning the basic mechanisms that regulate whether a synapse will form between two neurons. In fact much of our current thinking has arisen from the neuromuscular junction. The aims of the proposed research are to study synaptogenesis between neuron pairs and to investigate a specific synaptogenic regulatory mechanism. The hypothesis that "the formation of a chemical synapse requires the cessation of neurite extension in the presynaptic neuron" will be tested here. Using identified neurons of the pulmonate mollusc Helisoma it will be possible to gain a level of precision essential to such a study. Identified neurons can readily be plated into culture as specific neuron pairs where they will form previously characterized chemical synaptic connections. Furthermore, in cell culture it is possible to make direct experimental observations and manipulations. Thus, in testing this hypothesis, the timing of the cessation of neurite extension will be correlated with the timing of the formation of chemically synapses. Additionally, the cessation of neurite extension will be experimentally manipulated. The neurotransmitters serotonin and dopamine cause a premature cessation of neurite extension. Consequently these agents will be added to culture media to determine if synaptic connections form parrallel with our experimentally evoked inhibition of neurite extension. Such experiments take strides towards elucidating regulatory mechanisms controlling the formation of synaptic connections. To more directly investigate potential mechanisms that can regulate the formation of the synapse a higher level of resolution will also be utilized. In these studies single synaptic terminals will be voltage clamped at various times in the development of synaptic connections to determine whether the appearence of presynaptic ionic conductances may regulate the development of functional synaptic transmission. Thus, this proposal seeks to elucidate regulatory mechanisms that control the formation of chemical synapses, and to investigate such mechanisms at a new level of resolution by direct studies at single synaptic terminals.

calcium channel; G protein; neural transmission; synapses; neurotransmitter metabolism; exocytosis; calcium metabolism; tissue /cell culture; Gastropoda;

molecular biology; meeting /conference /symposium; microorganism toxin; virus cytopathogenic effect; vaccines; bacterial toxins; bacterial cytopathogenic effect; genetic transcription; exotoxins; virulence; saxitoxin; microorganism toxicology;

This award provides funds to aid in the purchase of a variety of equipment items to be used for biotechnology research by members of the zoology and biochemistry departments at Iowa State University. Equipment includes a state of the art imaging device for detection and analysis of radioactively-labelled nucleic acids and proteins, a device for densitometry of stained-gels and autoradiograms, equipment for synthesis and separation of DNA oligomers, a computer workstation for data analysis and several other items. The equipment will be housed in the nucleic acid facility of the new Biotechnology Center. Advances in basic research in Biotechnology, including studies in molecular biology and cell biology, have depended heavily on the development of instrumentation such as DNA synthesizers and sequencers that permit what had been time-consuming and difficult experimentation to be accomplished rapidly and with little effort. New devices have now increased the speed and sensitivity of detection and analysis of proteins and nucleic acids by orders of magnitude. Typically such equipment is placed in a central location where it is available to a large number of investigators who can benefit from its use. Precisely such an arrangement is proposed for the equipment to be purchased through this award.

Regulatory mechanisms controlling synaptogenesis are key to the normal development of the CNS. Much of our knowledge of synaptogenesis has been gained from the neuromuscular junction. This information has now permitted us to formulate hypotheses about the regulatory mechanisms controlling the formation of synaptic circuitry of mammalian central neurons. Using rat hippocampal neurons in cell culture we will elucidate regulatory mechanisms controlling the development of mammalian presynaptic nerve terminals. Knowledge gained in this study will be crucial for developing strategies to restore or repair damaged circuitry in the CNS. Specific Aim I: The developmental appearance of functional presynaptic calcium channels: The relative roles of intrinsic and extrinsic regulation. Using calcium imaging and patch clamp techniques on cell cultured hippocampal neurons, we will determine the developmental timetable for expression of functional calcium channels. We will determine effects of neuron-neuron contact on the expression of functional presynaptic calcium channels responsible for neurotransmitter release. Specific Aim II The role of glial-neuronal signaling in synaptogenesis. Electrophysiological evidence from our laboratory indicates that astrocytes regulate neuronal synapse development. We will ask which aspects of presynaptic development are regulated by astrocytes? Specific Aim III Is the rate-limiting step in synaptogenesis the expression of functional calcium channels or of functional secretory apparatus? By performing electrophysiology together with calcium imaging and flash photolysis of caged calcium (DM-Nitrophen) we will determine whether the rate-limiting step in synaptogenesis is due to the timing of expression of functional calcium channels or of functional calcium- regulated secretory apparatus. Specific Aim IV Signal transduction pathways controlling presynaptic development. Working from evidence obtained in peripheral synapses, we will now test the roles of cAMP and internal calcium, as well as other second messengers, as regulators of synaptogenesis of mammalian central neurons. This investigation will represent one of the first systematic cellular studies of synaptogenesis between mammalian central neurons. Using approaches that have been previously shown to be successful for peripheral neurons, we will obtain some of our first insights into the cell and molecular regulatory mechanisms which control the development of central presynaptic terminals.

During the past decade many candidate molecules have been identified that may play roles in regulating and triggering synaptic transmission. However, there have been limited opportunities to test the role of these proteins in the synapse because there are few tractable preparations where it is possible to integrate the necessary cellular and molecular approaches for the study protein function in the synapse. Using a system of synapses which form between spherical somata of Helisoma neurons, we are now in a unique position to utilize both cellular and molecular approaches in studying the synapse. We will distinguish between two roles for rab3 in the synapse; that it 1) controls vesicle docking with the plasma membrane; or 2) is a critical component of the vesicle fusion apparatus during calcium-dependent exocytosis. Molecular perturbation of rab3: immediate effects on synaptic transmission. Helisoma neurons form synaptic connections directly between apposed neuronal somata in culture. This permits the microinjection of macromolecules directly into the presynaptic terminal while monitoring the immediate effects on synaptic transmission. Using specific domain peptides, antibodies and chromophore-assisted laser inactivation (CALI), we will determine how perturbations of rab3 affect synaptic transmission. Molecular manipulation of rab3 expression: The requirement for rab3 in synapse function. We have recently demonstrated that we can effectively use antisense oligonucleotides to selectively prevent protein expression in Helisoma. Using this approach, we will ask whether the expression of rab3 is necessary for synaptic transmission. Expression of mutant rab3: Functional consequences for synaptic transmission. We have recently demonstrated that we can express proteins in Helisoma presynaptic neurons using a pNEX expression vector. We will use this expression system in presynaptic neurons to express mutant forms of rab3 that are impaired in their ability to bind GTP and will determine the consequences for synaptic transmission. We have known for decades that calcium, acting through a series of molecular events, stimulates neurotransmitter release. However, it has been difficult to precisely define this cascade of events. One essential protein is rab3, and by determining its role in the synapse this study will provide necessary information to put into place a critical building block in the emerging picture of the molecular events linking calcium to the release of neurotransmitter.

The primary goal of the proposed investigation is the detailed elucidation of the molecular aspects of toxin regulation in V. cholerae. Biochemical and genetic methods will be used to locate the DNA sites involved in formation of the cholera toxin operon (ctx) promoter and control sites. We will attempt to define the precise molecular mechanism by which the positive regulatory gene toxR, activates ctx transcription. The regulatory role played by the repetitive sequence TTTTGAT will be studied by localized and mismatch primer mutagenesis. The nutritional and physical factors which regulate toxin production will be correlated with specific transcriptional effects on ctx or toxR. We will continue to study ctx amplification as a genetic mechanism controlling toxin production in E1 Tor strains of V. cholerae. In this regard, we will test the repetitive sequence RS1 for its ability to mediate various recombinational events including transposition, duplication and site-specific recombination. The knowledge gained from all the above studies will be used to devise methods with which to study toxin regulation and ctx amplification in vivo. Specifically, we will study how the intestine provides a selective environment for increased toxin expression by defining which genes (ctxA, ctxB or toxR) are necessary for in vivo selection of genetic reversion and amplification events. We will apply what we have learned about ctx regulation and expression to the development of improved live and dead, oral vaccines against cholera. Mutations will be constructed in the cloned recA gene of V. cholerae and then recombined back in place of the rec+ gene of the prototype live oral vaccine strain 0395-N1. In addition to recA, streptomycin-dependent, DAP- and ga1E mutations will also be incorporated into 0395-N1 derivatives as a means of limiting the persistence of this strain in humans and the environment. Finally, derivatives of 0395-N1 which are suitable for use as a dead, oral cholera vaccines will be constructed which hyperproduce the B subunit of the toxin in both a secreted and nonsecreted form.

Near-field optical microscopy can provide a resolution greater than wavelength of light. In operation, light is passed down a tapered optical fiber and emerges at a fine tip whose diameter is smaller than the wavelength of light. This tip is brought into contact with a fluorescently labeled specimen and is scanned over the surface. Only a fluorophore in the immediate vicinity of the tip is maximally excited. However, there is a low level of excitation in the far field as the light disperses away from the tip. This low level background can be removed by confocal aperture in the imaging optics. We are exploring the alternative strategy of eliminating this background fluorescence by the use of multiple photon excitation. In this scheme, fluorophorr, excitation is proportional to the square or higher powers of the excitation illumination and therefore will essentially only occur in the immediate vicinity of the tip. We are proposing to investigate whether we can get sufficient levels of two-photon excitation in the vicinity of a subwavelength tip to enable images with a good signal to noise ratio to be obtained.

DESCRIPTION The overall goal of this proposal is to test the hypothesis that astrocytes modulate synaptic transmission. Previous work from the applicant's laboratory has shown that calcium elevations in astrocytes lead to delayed calcium elevation in adjacent neurons. In this proposal, the applicant will address three tightly interrelated hypothesis designed to examine the role of astrocytes. First, he will test the hypothesis that Ca++ dependent glutamate release from astrocytes requires the SNARE fusion complex. Second, he will test the hypothesis that activation of astrocytes causes mGluR and NMDA receptor dependent synaptic modulation. Finally, he will test the hypothesis that astrocytes regulate synaptic transmission by controlling intracellular calcium levels in presynaptic terminals. If successful, these experiments will provide solid information on the role of astrocytes in synaptic transmission.

Cellular calcium levels are one of a cells most responsive elements in many aspects of cellular regulation. In the astrocytes, which form the subject of this ongoing study, waves of calcium, initiated by mechanical stimuli or agonist, correlate with glutamate secretion. It is not known whether this release is vesicular in nature nor what calcium regulatory mechanisms are involved in the aftermath of the wave itself. Wave propagation is an IP3 dependent phenomenon and it is speculated that the rapid release of calcium from the endoplasmic reticulum is replenished by capacitative coupling through a calcium release activated current (ICRAC). The objective of this study was to investigate the interactions of the different calcium regulatory mechanisms and capitalize on the high sensitivity, microdomain analysis, of the self-referencing calcium probe. This technique was coupled to near-field optical fibres enabling cell specific release of caged calcium. An imaging system enabled the imaging of glutamate release via L-glutamate dehydrogenase and NADH fluorescence Still in progress the results of this study remain inconclusive. We have recorded weak steady-state calcium efflux signals from cultured confluent and single astrocytes. The signal, however, is not responsive to norepinephrine or cage calcium release, although both stimuli increase cellular free-calcium. Interestingly the efflux is eliminated in cells injected with either the caged calcium or BAPTA, suggesting that it is derived from transmembrane transport. Data indicate a response to mechanical stimuli but cellular variability still requires further analysis. Further experiments are planned to pursue the question of ICRAC contribution to the glutamate secretory response and its response to reticular calcium depletion. A different cellular model will be sought.

0088241
Porter
The objective of the proposed research is the design, development, and evaluation of a new paradigm for chip-scale bioanalytical laboratories. The goal is to uniquely couple the emerging fields of magnetically-based analyte labeling, DNA/protein microarray fabrication, microfluidic injection, and giant magnetoresistive sensors, forming a new class of integrated systems that address the enormous demand for high throughput sample screening in the functional genomics and proteomics arenas. These types of assays are also applicable to purity maintenance of public water and food supplies.

The goal of the FIRCA application is to study the molecular mechanisms of glutamate release from astrocytes. In the parent grant, the PI has shown that calcium signals in astrocytes cause the release of glutamate that in turn can modulate synaptic transmission. Additionally, it has been shown that SNARE proteins are necessary on the release of glutamate from these non-neuronal cells. Given the existing knowledge of the role of SNAREs in mediating excitocytosis, this leads to the hypothesis that exocytosis of glutamate filled vesicles underlies glutamate release from astrocytes. In order to test such a hypothesis it is necessary that the PI work with Dr. Robert Zorec (Slovenia) to measure the capacitance of astrocytes, an assay of exocytosis, a goal that is beyond the scope of the parent project. Through this collaboration four specific objectives will be addressed in this FIRCA project. Specific Aim I: Do elevations of intracellular calcium increase the membrane capacitance of astrocytes? Specific Aim II: Are SNARE proteins required for calcium-regulated changes in cell membrane capacitance? Specific Aim III: Do exocytotic events cause glutamate release from astrocytes? Specific Aim IV. We will determine whether we can detect exocytosis of a single vesicle from astrocytes. We expect that the results of this work, will contribute significantly to our understanding of whether glutamate release from astrocytes is mediated by regulated exocytosis. The clarification of the hypothesis that exocytosis is associated with glutamate release is of fundamental importance for the understanding of the role of astrocyte participation in neuronal plasticity.

[unreadable] DESCRIPTION (provided by applicant): During the past decade a surge of experimental evidence has shown that astrocytes release chemical transmitters which modulate synaptic transmission and neuronal excitability. In addition to taking up synaptically released neurotransmitters, astrocytes release glutamate, ATP and D-serine. Though it is now clear that astrocytes listen and talk to synapses, the functional implications of this bi-directional signaling pathway are not yet clear. During the past funding period we have generated astrocyte-specific inducible transgenic mice in which we can block the release of the chemical transmitter ATP from astrocytes in adult animals. With this mouse we have discovered that astrocytes coordinate synaptic networks by regulating the extracellular concentration of adenosine: astrocyte-released ATP is hydrolyzed to adenosine which suppresses the strength of excitatory synaptic transmission. The availability of this transgenic animal puts us in the unique position to test the following hypothesis. "Activity dependent release of ATP from astrocytes leads to the accumulation of extracellular adenosine that suppresses synaptic transmission neuronal excitability. The astrocyte-dependent accumulation of adenosine acts as a spatial filter, enhancing the contrast between neighboring synaptic pathways and limits the spread of excitation thereby suppressing the development of seizures." Using molecular genetics together with brain slice electrophysiology we will perform four specific aims of investigation: Aim I: We will test the hypothesis that the diacylglycerol arm of the phospholipase C (PLC) pathway is required to stimulate ATP release from astrocytes. Aim II: Synaptically stimulated release of purines from astrocytes causes a contrast-enhancement between neighboring active and inactive synapses. Aim III: Astrocyte-derived purines block the induction of LTP. Aim IV: By integrating the overall level of neuronal activity astrocytes have anti-convulsant actions through their control of extracellular adenosine. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Many diseases of the central nervous system (CNS) are characterized by the devastating loss of neurons. Several approaches have been investigated to determine whether they could either halt or reverse this neuronal loss including the introduction of neurotrophic factors as well as transplantation of neural stem cells to provide a source of new neurons for reinnervation of the CNS. Recently, adult mesenchymal stem cells (MSCs) have been shown to have the potential to take on neuronal and glial properties when treated with neurotrophic factors in cell culture. Moreover, our own preliminary studies have demonstrated that glial-derived neurotrophic factor (GDNF) treated adult MSCs gain a sensitivity to the neurotransmitter glutamate. These studies suggest that adult MSCs have the ability to take on neuronal and/or glial properties suggesting a potential avenue for treatment for certain neurological disorders: the patient could supply their own MSCs which, after treatment with appropriate neurotrophic factors in culture, could be transplanted into the CNS where they would have the potentia to differentiate into neurons and ameliorate the symptoms of the disorder. Using adult MSCs we will determine the mechanisms of GDNF-induced calcium signaling, and will test the hypothesis that GDNF induces neuronal differentiation of stem cells as assayed by calcium signaling in response to the neurotransmitter, glutamate. We will ask whether the GDNF-induced calcium signal is essential for neuronal differentiation and whether MSCs that are transplanted into the retina, morphologically and functionally integrate into the retinal circuitry. Using confocal imaging together with UV photolysis and photo-release of caged glutamate to probe the circuitry of the retina we will test the hypothesis that GDNF treatment of adult-derived mesenchymal stem cells causes neuronal differentiation, and the ability of MSCs to integrate into functional circuity of the CNS. By mechanistically determining the regulation of the neuronal glutamatergic phenotype we will be able to design rational approaches to differentiate adult MSCs along neuronal pathways with the long-term objective of gaining functional integration of these treated cells in the CNS.

Researchers are increasingly aware that astrocytes respond to neuronal activity with Ca2+ signals that can induce the release of chemical transmitters. The roles of these gliotransmitters in the control of neural function and behavior are poorly defined. Our studies have revealed that astrocytes are responsible for the control of extracellular adenosine that activates neuronal adenosine 1 receptors (A1R). The expression of a dominant negative dnSNARE domain in astrocytes, to inhibit the release of gliotransmitters, causes a reduction in the magnitude of CA3-CA1 long term potentiation (LTP)(Pascual et al., 2005) as well as a reduction in synaptic N- methyl-D-aspartate receptor (NMDAR) current (prelim studies) and impairments in sleep homeostasis. Since Ca2+ supplied by NMDARs is essential for the induction of LTP, we propose a novel hypothesis linking astrocyte-derived adenosine with NMDARs and LTP: Astrocyte-derived adenosine acting through A1 receptors enhances synaptic NMDAR currents and consequently the magnitude of NMDAR-dependent LTP. To test this hypothesis we will use conditional astrocyte-specific transgenic mice that allow both activation and inhibition of glial signaling pathways. We have four specific aims: First, we will test the hypothesis that astrocyte-derived adenosine acting on A1 receptors regulates synaptic NMDAR currents. Second, we will test the hypothesis that astrocytic Ca2+ signaling promotes NMDAR-dependent LTP. Third, we will test the hypothesis that astrocytic enhancement of LTP is mediated via A1 receptor-dependent augmentation of NMDA receptors. There are likely to be wide ranging effects of adenosine, NMDAR and LTP on behavior. To maintain focus we will build on our recent studies in the fourth specific aim to identify roles for astrocyte-Ca2+ signals and adenosine in the control of sleep homeostasis. This project will provide entirely new information on the role of astrocytes in brain function. Using molecular genetic studies in situ and in vivo we will determine under which conditions astrocytes contribute to information processing and behavior. Since we propose that astrocyte-derived signals regulate NMDA receptors, receptors known to be central to numerous disorders, this project has the potential to identify novel glial targets to enhance learning and memory and to treat sleep disorders.

DESCRIPTION (provided by applicant) Optical microscopy has been an essential technique in cell biology that has permitted the visualization of a variety of sub-cellular processes. When combined with selective labeling methods and fluorescent probes, optical microscopy has allowed the identification of the distribution of specific macromolecules and measurement of ion fluxes as well as the ability to monitor vesicle cycling in nerve terminals. However, because objective-based microscopy is limited by diffraction, the resolution of fluorescence microscopy is about 250 nm limiting studies to the behavior of populations of channels and vesicles. In this study we will apply biological near-field microscopy, with resolution of 20-50nm, to the study of the nerve terminal. Using this approach we will use high spatial resolution fluorescence microscopy to study the activity of single calcium channels and the behavior of individual vesicles in the nerve terminal. In order to achieve our overall objective we have two specific aims of study: Specific Aim I: We will use near-field optical fibers to map the microdomains of cytosolic calcium elevation during neuronal depolarization. Specific Aim II: We will determine whether second messengers that modulate synaptic transmission control the "mobility" of individual synaptic vesicles within the nerve terminal. Success in this project will have significant outcomes by allowing us to demonstrate the feasibility of a method that permits the application of sub-diffraction optics to living samples. In addition we will be able to perform the first investigation of the microdomains of the calcium ion, and will be able to address fundamental questions about synaptic transmission. With the data obtained from this study we will then have the preliminary data that are necessary to successfully compete for RO I style research grant applications.

The Consultation Core will fill an extremely important role in the Center; it will be the core responsible for advising investigators about the use of the Center's instruments for their studies. In particular, the Consultation Core will be essential for assisting users who have little experience with the microscopes available in the Center. Consultation will be free of charge and arranged through email or telephone. Each of the two Research Associates in the Center will dedicate 20% of their effort to consultation. They will be the initial contact persons for potential and established users and will discuss the experimental objectives of the investigator, give information about the capabilities of the instruments and suggest which instrumentation would be appropriate for a given study. Additionally, consultation can be sought after experiments are performed about methods of data analysis and interpretation. In addition to guiding investigators, the Consultation Core will serve the important task of screening potential studies to determine whether it will be appropriate to use the Center equipment for a given experiment. As discussed in section D2.2, we have selected members of our Steering Committee with diverse expertise, including electrophysiology, time-lapse imaging, calcium imaging confocal and two-photon microscopy as well as the use of voltage-sensitive dyes in physiological studies. The strengths of these individuals will be tapped by the Research Associates to assist in the consultation role of this core. Additionally, this core will send electronic newsletters to user groups. This newsletter will include items such as links to papers relevant to the use of the instrumentation in the users applications, as well as updates about new indicators and caged compounds, and information about the Center activities.

The Analysis core will house one workstation which will be PC-based and will contain the Zeiss analysis software and Image Pro. This instrument will be essential for the analysis of Zeiss Meta data in which linear unmixing of spectra is required. Once unmixed, however, users will be able to analyse their images with remote software in their own laboratories. In this way it will be possible to prevent potential bottlenecks of data analysis in the core facility. To achieve this objective we will purchase a 10-user site-license for Image Pro (in later years this will be upgraded to a 30-user license). With this site license up to 10 users will be able to simultaneously analyze their data. Zeiss has indicated their willingness to work with us to provide access to their image structure so that we will be able to directly load spectrallyunmixed Zeiss image stacks into Image Pro. Additionally, Zeiss permits the free download of a web browser analysis program. This does not have all of the features of the analysis software that will be loaded on the core workstation, but it will be a convenient supplement to Image Pro. Zeiss has told us of their plan to include the linear unmixing algorithm in the browser software. When this is included users will be able to perform all of their analysis at remote sites, as long as they copy the emission profile library of dyes that were collected on the microscope they used. We are aiming to have the majority of our users analyze their data at remote sites. This will reduce congestion in the core and reduce the total amount of space required by the Center. However, we will always maintain a local workstation in this core because it will permit the staff to work with individuals to guide them in methods of analysis. Additionally, this workstation will be used as part of the Consultation Core in assisting users in all phases of their studies. In addition to data analysis this core will also be responsible for managing two servers. The core will contain a server whose sole function is to perform weekly backup of computers within the Center. However, data backup will not be performed on primary data as this will be the responsibility of the user. The second server will be a data server which will act as a convenient site for users to port their data at the end of an experiment. Because of the amount of data that can be collected in an imaging experiment, and because of the time that it takes to write data to portable media, the user will be responsible for sending data from the acquisition instrument to the data server at the end of an experiment and then deleting the data files from the user-accessible portion of the instrument. Subsequently, the user will either write the data to removable media at this location or will transfer the data to a remote computer. All users will be required to delete their files within three days of transfer to this location. If they do not, the computer will send a reminder message which, if ignored, will result in the core staff deleting the data on the subsequent day.

We will purchase two Zeiss 510 Meta confocal microscopes for our studies. These microscopes will be configured on fixed-stage upright microscopes and will come with 458, 477, 488, 5t4, 543 and 633 nm laser lines, two internal photomultiplier tubes and a Meta, 32 element spectral detector. The 510 Meta confocal microscope is the instrument of choice for studies because of its ability to detect emission spectra and to unmix overlapping spectra of multiple indicators while performing the high-quality confocal imaging expected of a 510 system. We feel that the Meta detector is a revolutionary concept in microscopy which will significantly facilitate our biological studies; it will permit our investigators to simultaneously express multiple fluorescent proteins in cells and, despite their similarity in emission spectra, to quantitatively identify the contribution of each. With 20 investigators and 34 NINDS projects in this proposed Center, each investigator would have only two hours per week (or each N1NDS project would have just over one hour per week) on the microscope were we to have only one two photon microscope available. Thus the purchase of two of these two-photon microscopes is essential. Because CHOP appreciates the great expense of these microscopes, it has provided $259,835 to alleviate some of the financial burden.

It is often averred in the Life Sciences that we are not short of questions; rather, we are limited by the availability of technologies to address these questions. During the past decade several new technologies have been developed to address biological problems. However, barriers to using these newly-developed technologies are often considerable for the individual investigator. The goal of this Center is to provide a centralized and staffed facility to allow both the expert and the novice to use the most modern research tools in their studies, and to equip these instruments for dynamic imaging in physiological studies. We will develop a Center around three cores Dynamic Imaging Core, containing, two two-photon microscopes, spectral detection confocal microscopes, total internal reflectance fluorescence microscope together with photolysis attachments; Data Analysis Core that will provide opportunities for off-line analysis of all data collected with the Center's microscopes, and Consultation Core for new and established users of the Center's Dynamic Imaging Core. The goals of this Center fall into three scientific clusters: I) Neuronal Development, in which ot projects will investigate problems ranging from stem cell biology to neuronal migration and synapse formation, lI) Regulation of Ion Channels and Synaptie Transmission, in which our studies include protein-protein interactions controlling calcium channel activity, the spatial distribution of receptors, and the localization of synapses controlling neuronal networks, and Ill) Gila-Neuron Interactions, in which we will investigate calcium signaling in astrocytes, the roles of ion channels in myelination, and the role of glial-released messengers in synapse formation. Establishing this Dynamic Imaging Center will catalyze new studies and augment existing studies in the 21 qualifying N1NDS-funded laboratories (including 34 N1NDS grants) that comprise this Center. The resultant synergistic interactions between faculty members will revolutionize our ability to study basic functions of the nervous system function and will ultimately enhance our understanding of these functions in human health and disease.

synaptotagmin; neuroregulation; neurotransmitter transport; astrocytes; protein structure function; synapses; glutamates; photolysis; pyramidal cells; messenger RNA; neural plasticity; calcium flux; hippocampus; tissue /cell culture; genetically modified animals; laboratory mouse; electroporation;

DESCRIPTION (provided by applicant): During the past decade there has been a significant change in our view of synaptic function: it has now become apparent that the synaptically associated astrocyte, is key for the control of synaptic transmission. Consequently, a concept of tripartite synaptic transmission has been proposed. Though the concept has been formulated, we are at the very early days in our understanding of the relative roles of presynaptic, postsynaptic and astrocytic elements in the control of functional synaptic transmission. The goal of this Conte center is to bring together a team of talented and experienced investigators to focus collaborative studies on the tripartite synapse. Our goal is to change the landscape of thinking by testing the overriding hypothesis that "Bidirectional signaling between astrocytes and pre- and postsynaptic neurons regulates the function of the tripartite synapse". To address this hypothesis we propose four collaborative projects: Project #1 (Coulter, project director), will study the role of astrocytes in regulating the loading of vesicles with transmitter by controlling the recycling of neurotransmitter substrates. Project #2 (Eberwine, project director) will take a longer-term view of the astrocyte by studying the gene expression profiles of astrocytes, and investigate transmitter-dependent regulation of translation within astrocytes and their processes. Project #3 (Haydon, project director) will study the role of synaptotagmin IV in releasing gliotransmitters from astrocytes and how the short and long-term regulation of this protein controls synaptic transmission and plasticity. Because imaging and photolysis will be critical for many of the studies performed in this Conte Center we will include a photochemistry core (Ellis-Davies) within the center that will provide and develop new caged compounds for photo-release by two-photon photolysis. Collectively these projects will dramatically enhance our understanding of the tripartite synapse leading to new insights into the control of the functional circuitry of the brain.

DESCRIPTION (provided by applicant): Although stem cell/progenitor transplants promise unrivalled opportunities to repair damaged and aging nervous systems, success with this approach will only become possible after identification of the regulatory principles that control the functional differentiation of transplanted cells in the nervous system. Currently the technical approaches available to studying progenitor cell differentiation limit our ability to advance this field. Ideally one would repeatedly use non-invasive imaging approaches to whole animals to monitor the functional differentiation of transplanted cells. Unfortunately, available techniques such as MRI, that offer non-invasive imaging, do not provide the necessary spatial resolution. In this project we will develop a new approach in which we integrate two-photon imaging with two-photon photolysis to monitor and probe transplanted progenitor cell differentiation. To achieve this overall goal we have three objectives. First, we will refine optical stimulation and recording approaches that permit us to study the functional activity of transplanted progenitors. Using a genetically encoded Ca2+ indicator together with two-photon FRET microscopy and photolysis we will develop imaging methods that allow us to study their integration into host circuitry. Second, we will develop repeated in vivo imaging strategies that employ non-invasive two-photon imaging and photolysis to track the functional differentiation of transplanted progenitors in vivo for periods of up to one year. Finally, we will employ these imaging strategies to determine the relative merit of differentiating progenitors by exposure to growth factors either in vitro prior to or in vivo subsequent to transplantation. The development of vital imaging methods to assess progenitor cell differentiation will provide a new approach to allow the identification of the mechanisms which positively regulate differentiation and integration of transplanted progenitors. Since we will be able to assess outcomes based on cellular function that will surely be detected before behavioral consequences can be observed, the successful development of this imaging approach will accelerate the pace of research with behavioral outcomes goals.

Though much work has focused to synaptic changes in the nervous system that follow injury and that underlie seizure disorders, the role of glial cells in the disorder have largely been speculative. Status epilepticus, a period of repeated often intractable seizures, is followed by a latent period of epileptogenesis during which there is a delayed death of neurons in the limbic system and cellular re-organizations that lead to the acquisition of spontaneous seizures (epilepsy). We know that astrocytes become reactive following status epilepticus but how they influence neuronal function is unknown. Our previous physiological studies demonstrated that Ca2+ elevations in astrocytes evoke the release of glutamate from these glial cells (gliotransmission) which in turn regulates neuronal excitability. Recent preliminary studies, employing in vivo two-photon Ca2+ imaging, demonstrate that astrocytes exhibit a prolonged period of enhanced Ca2+ excitability following status epilepticus. Since cell-wide glial Ca2+ oscillations evoke NMDA-receptor dependent neuronal excitation that is synchronous amongst several pyramidal neurons, we propose that astrocytes contribute to the regulation of neuronal excitability and excitotoxicity following status epilepticus. By integrating two-photon microscopy and photolysis with electrophysiology and inducible cell-specific transgenic animals we will determine the relative roles of gliotransmission in the regulation of neuronal excitability and excitotoxicity. We have three aims in our project. We will test the hyptheses that: I. Status Epilepticus evokes increased astrocytic Ca2+ oscillation frequency lasting for 1-3 days that stimulates glial glutamate release. II: Ca2+ oscillations in astrocytes evoke NMDA-mediated excitation of neurons during the period of enhanced astrocyte excitability that follows status epilepticus, and III: Ca2+-dependent gliotransmission induces neuronal death by the activation of NR2B-containing NMDA receptors. Though there has been speculation about the potential role of astrocytes in epileptogenesis and epilepsy, an absence of an understanding of the properties of these glial cells together with the virtual absence of astrocyte-specific experimental manipulations has prevented an appreciation of the role of these glia in epileptogenesis. This study will shed new light on and how astrocytes contribute to epileptogenesis and will provide new insights into the generation of epilepsy that have the potential to provide a new therapeutic target for the prevention of epileptogenesis.

The calcium-binding synaptotagmin family of vesicle proteins control vesicle fusion with the plasma membrane. The best studied family member, synaptotagmin I, is necessary for fast, synchronized neurotransmitter release from presynaptic terminals. While the roles of synaptotagmin I are being elucidated, the roles of the additional 14 members of the group are less clear. However, our preliminary studies support the notion that astrocytes, but not hippocampal pyramidal neurons, express synaptotagmin IV. Given that synaptotagmin IV knockout mice have demonstrated deficits in hippocampal-based learning and memory, the goal of project 3 is to test the hypothesis that synaptotagmin IV is essential for calcium-triggered exocytotic release of glutamate from astrocytes and that this regulated transmitter release pathway is critical for the control of synaptic transmission at the tripartite synapse. In the first aim we will test the hypothesis that astrocytic synaptotagrnin IV regulates glial transmitter release and the modulation of synapses in situ (Haydon, Ellis-Davies and Coulter). It is well established that trains of synaptic activity lead to long-term modifications of the synapse. We hypothesize that such activity similarly causes a long-term change in the astrocytic arm of the tripartite synapse. In collaboration with project 2, we will, in the second aim, ask whether synaptic activity regulates the synthesis of gliotransmitter release machinery within astrocytes in situ (Haydon, Ellis-Davies and Eberwine). The molecules studied in this experiment will be dependent on the initial results from project 2 where mRNAs resident within astrocytes and those specifically within glial processes will be identified. However, one candidate that we will study is synaptotagmin IV. To achieve this objective we will build on our recently developed ability to use electroporation to introduce mRNA into astrocytes in acutely isolated hippocampal slices. This aim is not feasible in a stand-alone project and will require close interactions as the outcomes of project 3 will dictate the directions taken here. By elucidating control functions of astrocytes over synaptic transmission this collaborative Conte Center project has the potential to change the way we view both short- and long-term regulation of the synapse.

DESCRIPTION (provided by applicant): Synaptic plasticity is at least one of the cellular underpinnings of addiction to drugs of abuse. NMDA receptors, which are necessary for some forms of synaptic plasticity, play pivotal roles in mediating behavioral responses to cocaine. Infusion of cocaine can lead to NMDA receptor-dependent long term potentiation of synaptic transmission in the ventral tegmental area (VTA). Infusion of NMDA receptor antagonists into the VTA prevent cocaine-induced conditioned place preference. We will test the novel hypothesis that astrocytes are critical for the control of NMDA receptor function, synaptic plasticity, and as a consequence addictive behaviors. There is a new appreciation for roles of astrocytes in the control of synaptic transmission. In 1994 we discovered that astrocytic Ca2+ signals stimulate the release chemical transmitters from these glia. Since then we and others have shown that this process of gliotransmission can regulate neuronal excitability and synaptic transmission leading to the idea of the Tripartite Synapse, which accounts for roles of astrocytes in synaptic transmission. Using lines of inducible, astrocyte-specific transgenic mice impaired in gliotransmission we have made two observations essential for this project: First, inhibiting gliotransmission significantly reduces synaptic NMDA receptor density. Second, this inhibition of gliotransmission blunts cocaine-induced conditioned place preference. Given the known importance of NMDA receptors in mediating rewarding properties of drugs of abuse we hypothesize that astrocytes regulate neuronal NMDA receptor density and synaptic plasticity and thereby behavioral responses to drugs of abuse. Specific Aim I: Test the hypothesis that gliotransmission regulates functional NMDA receptor density on dopaminergic neurons in the VTA. Specific Aim II: Test the hypothesis that gliotransmission promotes synaptic plasticity in the VTA. Specific Aim III: Test the hypothesis that gliotransmission is essential for cocaine-induced behavioral response. Systematically evaluating the role of gliotransmission in synaptic plasticity and behavioral responses to drugs of abuse promises to offer new insights into the cellular mechanisms underlying addiction. Since astrocytes express unique receptors that could be targeted therapeutically, success in this project may offer a new approach to prevent and treat addictions. PUBLIC HEALTH RELEVANCE: The goal of this work is to evaluate the novel idea that a non-neuronal cell of the brain called the astrocyte plays a pivotal role in mediating the long-term response to drugs of abuse. Systematically evaluating the role of astrocytes in synaptic plasticity and behavioral responses to drugs of abuse promises to offer new insights into the cellular mechanisms underlying addiction and the potential to identify new targets for the development of therapeutics.

This project is for partial support for an international meeting on Glial Biology, as part of the Gordon Research Conference series, to be held in Ventura, California, March 15-20, 2009. The broad and long-term goal of the conference is to increase our understanding of the manner in which glial cells in the brain interact with one another and with neurons during normal brain function and in neurological diseases. The biology of two specific glial cell types, astrocytes and microglial cells, is emphasized in this meeting. The specific aim of this meeting will be to convene 44 speakers to discuss critical areas of glial research with a total of 140 participants, during a five-day conference. The program will address glia cell involvement in synaptic structure and function, neurogenesis, brain energy metabolism, cerebral blood flow, and some neurological diseases including tumors and stroke. The Glial Biology GCR is designed to: 1. Initiate and help sustain an interactive dialogue between scientists in different fields of glial biology and glial-neuron interactions by providing a long-term forum for presentation of cutting-edge work to a diverse group of leading researchers. 2. Suggest new approaches to solve timely glial biology problems and help identify controversial issues in this field that require resolution. 3. Raise awareness in the neuroscience community of the importance of glial-neuronal interactions. Specific presentations at this conference will focus on the involvement of glial cells in brain development, tumor formation and spread, normal and deranged energy metabolism (e.g., stroke and hypoglycemia) and pain perception. Students and post-doctoral fellows will be invited and will have a unique opportunity to learn from some of the most distinguished glial scientists in the world.

DESCRIPTION (provided by applicant): The Neurosciences at Tufts University School of Medicine (TUSM) have recently been identified, as part of a Strategic planning process, for expansion by hiring a new chair and new faculty. The PI of this application has been recruited to Tufts as the Chair of the Neuroscience Department and has already made four additional hires. The purpose of this application is to form a partnership between the NIH and Tufts so that we can expand this program to hire additional tenure-track assistant professors. We request funds to hire two tenure-track assistant professors, and together with funds from TUSM provide the setup packages for this new faculty. This institutional commitment includes the recent renovation of new laboratory space for these recruits (completed in May 2009), $1.4 million of funds towards the total setup package, as well as a guarantee of hard salary support for the recruits for the two year period beyond the completion of this grant. The Neurosciences at TUSM is focusing on three specialties to enhance our Synapse Group: the study of synaptic transmission per se, as well as studies of disorders of the nervous system and neuron-glial interactions within the context of the synapse. Each of these areas is mutually supportive and partially overlapping providing focus to our development. Moreover, this blend of specialties provides us with our own niche so that as we excel we can be recognized as leaders in these areas. In this project we have three objectives: Objective 1: Recruit the best candidates to enhance the study of synapse neurobiology at Tufts. Objective 2: IV Ientor, enhance and promote the careers of newly recruited faculty. Objective 3: Augment collaborative studies at Tufts through Core Research Grants. By achieving these objectives our goal is to develop a world-class program that is performing state-of-the-art studies in synapse neurobiology and is identifying the mechanisms underlying the control of brain and behavior in health and disease.

DESCRIPTION (provided by applicant): This proposal requests partial support for an international meeting on Glial Biology, as part of the Gordon Research Conference series, to be held in Ventura, California, March 6-11, 2011. The broad and long-term goal of the conference is to increase our understanding of the manner in which glial cells interact with one another and with neurons during normal brain function and in neurological diseases. The biology of astrocytes and microglial cells, more than that of oligodendrocytes, is emphasized in this meeting. The specific aim of this meeting will be to convene 44 speakers to discuss critical areas of glial research with a total of 150 participants, during a five-day conference in a relatively isolated setting. The program will have nine sessions that broadly address glia cell involvement in synaptic structure and function, synapse development, cerebral blood flow, and some neurological diseases including Epilepsy Alzheimer's disease and ALS. In addition, two poster sessions will permit all participants to contribute to discussion of these topics. The significance of this application is that the Gordon Research Conference on Glial Biology is an essential component of the recurring conferences that promote worldwide research on glial cells, and help define critical research areas in need of experimental resolution. The health relatedness of this application is that glial cells participate in many, possibly all, neurological diseases. Discussions about current research will advance our understanding of glial cells in both health and disease. In addition, specific presentations at this conference will focus on the involvement of glial cells in brain development, epilepsy, Alzheimer's disease, ALS, and pain perception. PUBLIC HEALTH RELEVANCE: The health relatedness of this application is that glial cells participate in many, possibly all, neurological diseases. Discussions about current research will advance our understanding of glial cells in both health and disease. In addition, specific presentations at this conference will focus on the involvement of glial cells in brain development, epilepsy, ALS, Alzheimer's disease, pain perception. By helping to define key questions that require experimental resolution we can advance our understanding of how glial cells are involved in neurological diseases, which is the first necessary step to lessening the burden of these diseases on society. Disclaimer: Please note that the following critiques were prepared by the reviewers prior to the Study Section meeting and are provided in an essentially unedited form. While there is opportunity for the reviewers to update or revise their written evaluation, based upon the group's discussion, there is no guarantee that individual critiques have been updated subsequent to the discussion at the meeting. Therefore, the critiques may not fully reflect the final opinions of the individual reviewers at the close of group discussion or the final majority opinion of the group. Thus the Resume and Summary of Discussion is the final word on what the reviewers actually considered critical at the meeting.

DESCRIPTION (provided by applicant): A serious global public health problem concerns maintaining the quality and effectiveness of life as people live longer and are thus susceptible to more chronic and acute diseases and disorders. Recognizing this opportunity, the current administration of Tufts University has invested in the interdisciplinary area of Neurosciences research and education with the goal of developing this field into one of its leading programs. At the heart of this plan is the identification of the behavioral consequences of molecular genetic manipulations with the long term goal of identifying new therapeutic targets for the treatment of disorders such as epilepsy, depression, disorders of learning, memory and cognition, as well as neurodegenerative disorders. Such studies require a detailed analysis of murine behavior, and thus access to behavioral testing facilities is essential for contemporary translational neuroscience research. At Tufts University School of Medicine (TUSM), our behavioral testing facility is an integral component of our Division of Laboratory Animal Medicine (DLAM). However, the behavioral facility of DLAM no longer has the capacity required to support the research of our growing research community. We request funds to expand the murine behavioral facility within DLAM. Our goal is to provide the Neuroscience research community at Tufts with a shared facility totaling 1,753 square feet (a 1.78-fold increase over the current facility) that provides a necessary range of advanced state- of-the-art behavioral testing services in order to accelerate high-impact research. Aim 1: Enlarge the Animal Behavior Facility within the existing Division of Laboratory Animal Medicine (DLAM) facilities. We will expand the Behavior Core into adjacent DLAM space, resulting in a doubling of the number of testing rooms for studying murine behavior. We will also expand the cage capacity of a reverse light cycle mouse holding room that is located in the facility while maintaining additional facilities that include a procedure room for surgery. The behavior core expansion will leverage additional funds from TUSM for the renovation of new space for DLAM that will yield a net increase in cage capacity. Aim 2: Provide adequate capacity and facilities for advanced behavioral testing modules and animal holding rooms. Because of limited space within the existing behavior core, much of behavioral testing equipment sits in storage and needs to be shuttled in and out of the behavior core to accommodate each investigator's needs. The expansion of the core will allow us to develop individual testing rooms targeted to specific behavioral modalities and will allow us to accommodate this equipment on a full time basis within the core. Support from the NCRR together with other investments provided by Tufts University, will allow a partnership that allows dramatic breakthroughs in our understanding of disorders of the brain, including epilepsy, depression, schizophrenia and Alzheimer's disease.

DESCRIPTION (provided by applicant): Alcohol has numerous actions in the nervous system mediated through multiple transmitter signaling pathways. Both acute and chronic actions of alcohol modify sleep related behaviors through undefined mechanisms. We have shown that astrocytes, a type of glial cell, modulate sleep homeostasis by an adenosine receptor 1 (A1R)-dependent mechanism. We have novel evidence that molecular genetic manipulations directed at this glial pathway also impact alcohol-induced behaviors. We propose that alcohol activates the adenosine- dependent astrocytic cell and molecular signaling pathway that normally contributes to the homeostatic drive to sleep. Our overriding hypothesis is that an astrocytic source of adenosine mediates behavioral sensitivity to alcohol, and that the comorbidity of alcoholism and sleep disruptions involves long-term perturbations of this adenosine pathway. This project will be divided into three sections. Initially, we will identify the astrocyte-based signaling pathways that contribute to acute effects of alcohol on behavior (Aim 1). Subsequently, we will study how pre-existing impairments in sleep homeostasis impact alcohol behaviors (Aim 2), and how chronic alcohol exposure modifies sleep homeostasis (Aim 3). Despite numerous clinical reports that emphasize the correlation between sleep homeostasis and alcohol behaviors, few experimental models have been developed to explore this relationship. The significance of this project is reflected in the development of novel experimental models and multiple techniques that will be used to identify the interaction between alcohol behaviors and sleep impairments. The approaches described below offer distinct opportunities for therapeutic intervention. The proposed study uses an innovative integration of multidisciplinary approaches to study the involvement of the astrocyte-dependent sleep homeostat as a key mediator of acute and chronic effects of alcohol. Since astrocytes are now known to express G protein coupled receptors that are not expressed in neurons, results may also enhance the future potential to identify novel targets for ameliorating sleep impairments that haunt recovering alcoholics.

DESCRIPTION (provided by applicant): Sleep abnormalities are co-morbid with many psychiatric conditions though whether sleep disorders are a cause or consequence of depression is unclear. A total night of sleep deprivation has immediate antidepressive actions in the clinical population, although the signaling pathway is unknown. We propose that adenosine underlies the antidepressive effects of sleep deprivation because i) adenosine regulates sleep, ii) sleep deprivation elevates adenosine, and iii) single nucleotide polymorphisms in adenosine transporters and a metabolic enzyme have been identified in patients with depression with disturbed sleep. We demonstrated that astrocytes contribute to the behavioral responses to acute sleep deprivation. We conditionally expressed the SNARE domain of a vesicle protein in astrocytes to impair exocytosis resulting in reduced extracellular adenosine, as assessed by reduced basal activation of neuronal A1 receptors (A1R), reduced slow wave activity (SWA) during non-rapid eye movement (NREM) sleep and impaired recovery sleep following sleep deprivation. We hypothesize that the antidepressive effects of acute sleep deprivation are mediated by astrocyte-derived adenosine acting on neuronal A1 receptors. Aim 1: We will test the hypothesis that astrocytic modulation of sleep homeostasis contributes to antidepressive effects of sleep deprivation. We will determine whether a total night (12h) of sleep deprivation leads to antidepressive like effects, then using dnSNARE mice will ask whether the astrocytic sleep homeostat is required to mediate depressive-like responses. Aim 2: We will test the hypothesis that A1 receptors are required to mediate the antidepressive-like effects of sleep deprivation. We will determine whether 12h of sleep deprivation leads to enhanced activation of A1R and using central delivery of A1R antagonists and A1R knockout (A{1}R[-/-]) mice we will determine whether antidepressive-like effects of sleep deprivation require A1R. Aim 3: We will test the hypothesis that sustained (12h) pharmacological activation of central A1R will produce antidepressive like effects. We will deliver A1R agonists intracerebroventricularly (i.c.v.) and wil ask whether the activation of this receptor pathway yields antidepressive like effects. Aim 4: We will determine the role of frontal cortex in contributing to antidepressive effects of sleep deprivation. We will expand preliminary c-fos immunoreactivity studies and the use of novel Tet Tag transgenic mouse that enables GFP-labeling of c-fos active neurons to identify sleep deprivation activated neurons of the frontal cortex. Finally, we will virally transduce frontal corex astrocytes with dnSNARE and ask whether region specific transduction inhibits antidepressive effects of sleep deprivation. The identification of a signaling pathway underlying antidepressive like effects of sleep deprivation has the potential to for the future development of glial-based therapeutics for the immediate relief of depression. PUBLIC HEALTH RELEVANCE: We will test the hypothesis that the antidepressive effects of sleep deprivation are mediated by glial cells signaling to adenosine A1 receptors. The identification of a signaling pathway underlying antidepressive like effects of sleep deprivation has the potential to identify a new therapeutic area for immediate relief of depression.

There is a strong comorbidity of narcolepsy and diabetes/obesity; however, the causal underlying mechanism is unclear. We recently performed studies using astrocyte-specific connexin 43 (Cx43) knockout mice (Cx43 KO) and found that they display both a narcolepsy-like phenotype and metabolic dysregulation. These linked phenotypes, arising from a single genetic manipulation, raise the potential that we have identified a cellular and molecular underpinning of this clinical linkage. We will use the collective strengths of Drs. Haydon and Kong, who are highly experienced in studying astrocytes and the control of sleep/wake cycles (Haydon) and the study of the hypothalamic neural circuits and metabolic control (Kong). Together, we will test the hypothesis that astrocytic connexins are essential for the supply of lactate as an energy substrate to orexinergic neurons, and in doing so, modulate orexinergic control of wakefulness and metabolic control. Pierre Magistretti and colleagues proposed an attractive hypothesis concerning metabolic coupling between astrocytes and neurons in which the astrocyte metabolizes glucose to lactate, then shuttle this energy source to neurons where lactate is converted to pyruvate, which is used in oxidative phosphorylation. This Astrocyte- Neuron Lactate Shuttle (ANLS) is attractive because: i) astrocytes contact the vasculature and express GLUT1, a glucose transporter, they can take up glucose. ii), astrocytes are considered to be biased towards glycolysis, and iii) neurons express monocarboxylate transporters (MCT) that are required for the uptake of lactate. We will extend our initial observations to test the hypothesis that astrocyte-derived lactate is required by orexinergic neurons to promote their electrical activity and that experimental manipulation of orexinergic neuronal activity is both necessary and sufficient to cause narcolepsy and metabolic disorders. Aim I: We will test the hypothesis that the deletion of Cx30 and Cx43 impairs the Astrocyte-Neuron Lactate Shuttle AND promotes narcolepsy and systemic metabolic dysfunction. Aim II: We will test the hypothesis that astrocyte-derived lactate modulates orexinergic neuron activity. Aim III: We will test the hypothesis that the activation of orexinergic neurons is sufficient to rescue normal phenotypes in connexin KO mice.

ABSTRACT Alzheimer?s disease (AD) is a progressive neurodegenerative disorder characterized by memory impairments and cognitive deterioration. Aging is the major risk factor for AD. Furthermore, increasing evidence indicates that astrocytes and microglia are implicated in the pathogenesis of AD. The ?4 allele of the apolipoprotein E gene (APOE) has been identified as a major risk factor contributing to the pathogenesis of sporadic AD (SAD) in about 15-20% of the cases. APOE is the major apolipoprotein expressed in the human brain primarily by astrocytes and to a lesser extent by microglia, and is involved in cholesterol homeostasis, and regulates A? clearance. Furthermore, genome-wide association studies (GWAS) have identified polymorphisms in genes enriched in microglia (e.g. SORL1, CR1, CD2AP, CD33, TREM2, ABCA7) and astrocytes (e.g. CLU and ABCA7) that increase the risk of developing AD. Recent advances in stem cell technology have allowed the reprogramming of primary cells from human subjects into induced pluripotent stem cells (iPSCs) and their differentiation in neurons, astrocytes and microglia. However, conventional 2D culture systems fail to recapitulate the diversity and maturation of multiple cell types and their interaction under physiological and pathological conditions. To overcome these weaknesses we have developed a novel bioengineered model of iPSC-derived neural tissue. Our silk-collagen protein-based ?donut? scaffolds can support compartmentalized, 3D brain-like tissues over a year, without necrosis. This tissue model is highly innovative, supporting the differentiating neurons growth in a donut-shaped porous silk sponge within an optically cleared collagen-filled central region for axon connectivity and synapse formation, that will allow for the first time live in vivo studies (e.g., cell-based electrophysiology, trafficking, synaptic functionality) of an human AD brain-like tissue during ageing (months of cultivation) under controlled experimental conditions. More importantly, the architecture of the scaffolds was optimized to meet the metabolic demand of high-density cell cultures in terms of free diffusion of nutrients and oxygen, a fundamental requisite for long-term cultures and ageing-related studies. Thus, we propose to: 1) Assess genotype-phenotype relationship of AD genetic variants enriched in astrocytes and microglia in patient-derived 3D brain-like cultures; 2) Assess genotype-phenotype relationship of AD genetic variants in vivo after transplantation of patient-derived cells in mice.

We propose that sleep disturbances of Alzheimer's disease (AD) result from a reduction in the delivery of energy substrates from astrocytes to neurons. In particular, the focus of this project is the reduction in Slow Wave Activity (SWA) in non-rapid eye movement (NREM) sleep that is present in patients with AD (Lucey et al. 2019). Since the power of SWA is proportional to the drive or pressure to sleep, reduced SWA will result in fragmented and poor-quality sleep. Understanding what causes this reduced SWA will provide key insights into early disturbances in patients with AD. In our preliminary studies we have identified that a mouse model of AD displays this characteristic reduction in SWA and we hypothesize that hypo-glucose metabolism in AD leads to reduced supply of lactate from astrocytes to neurons which in turn causes a reduction in pyramidal neuron excitability and consequently to reduced power of SWA in NREM sleep. Our previous studies have shown that astrocyte-derived lactate, supplied by glial glycolysis, is shuttled to neurons where it is converted to pyruvate as an energy substrate. If lactate supply is depleted, via deletion of astrocytic connexin 43, and consequently neuronal pyruvate is decreased, a neuronal KATP channel is activated, hyperpolarizing and silencing neurons (Clasadonte et al. 2017): in the lateral hypothalamus this results in quiescence of orexinergic neurons that require activity to drive sustained wakefulness, and in the cortex reduced pyramidal neuron activity leads to a decrease in the power of SWA in NREM sleep (Figure 3). Since it is known that there is hypo-metabolism of glucose in AD we therefore propose that this leads to a reduced supply of astrocyte-derived lactate and impairment of neighboring neurons that rely on this energy substrate. In this R21 project we will bring together the tools for the study of the astrocyte-neuron lactate shuttle (ANLS) with expertise in studying Alzheimer's disease and sleep to test the hypothesis that a reduction in ANLS leads to reduced power of SWA in NREM sleep, a phenotype of patients with AD. We will test the following hypotheses: Aim I: Alzheimer's mouse models show reduced power of SWA in NREM sleep. Aim II: We will test the hypothesis that changes in extracellular lactate are diminished in AD mice during sleep/wake transitions. Aim III: Deletion of monocarboxylate transporters reduces lactate transport and AD sleep/wake related phenotypes? We are submitting this project as an R21 since it represents a new area of study and though we demonstrate the feasibility of all approaches, we need to integrate several different experimental strategies to begin to test the hypothesis. Success in this project would then provide the preliminary data for subsequent RO1 applications studying the control of sleep in Alzheimer's disease.