Darwin K. Berg - US grants

The functions of nicotinic acetylcholine receptors (AChRs) in the nervous system are almost completely unknown. One of the most abundant AChRs in both the CNS and PNS is a species that binds alpha-bungarotoxin (alphaBgt), contains alpha7 subunits, and has a high relative permeability to calcium. This proposal examines the function of such receptors (alphaBgt-AChRs) in a simple vertebrate neural circuit. The experiments use whole-cell patch clamp on neurons in situ to test the hypothesis that alphaBgt-AChRs generate most of the synaptic current despite having a perisynaptic location. Patch clamp recording from presynaptic calyces will be used to test the hypothesis that presynaptic alphaBgt-AChRs also are present and modulate transmitter release. Subunit-specific monoclonal antibodies (mAbs) will be used to identify presynaptic AChR species, determine their subunit composition, and visualize their locations via confocal immunofluorescence. Sharp electrode intracellular recording-will be used to determine whether alphaBgt-AChRs increase synaptic efficacy and support high frequency transmission through the ganglion. Application of alphaBgt in vivo, together with surgical manipulations, will be used to determine whether the receptors alter neuronal survival, synaptic development, or receptor distribution during embryogenesis. A major goal will be the development and use of strategies for manipulating the levels of neuronal AChR gene products in vivo and in cell culture. The approaches will include transient transfection of postmitotic neurons in culture and retroviral infection of embryos in vivo as two vehicles for introducing epitope- tagged mutant constructs, dominant negatives, and targeted ribozymes. The strategies will be employed to alter patterns of AChR expression and discover receptor roles in the development and function of identified neural circuits. The biomedical relevance of neuronal AChRs is clear: they must participate in the powerful addictive quality of nicotine responsible for vast numbers of chronic smokers world-wide. Neuronal AChRs have also been implicated in a range of brain functions including state of arousal, level of attention, and short-term memory formation. AChR changes have been reported in patients suffering from Alzheimer's disease, Parkinson's disease, and other cognitive and behavioral disorders. The studies outlined here represent basic research intended to illuminate the role of nicotinic receptors in the nervous system. More broadly, they may provide new insight into fundamental aspects of synaptic function and lay groundwork for considering therapeutic strategies involving nicotinic receptors.

Bonner Hall, constructed in 1965, is one of the major buildings that houses the Biology Department at the University of California, San Diego. It is also one of the oldest buildings on campus and is overdue for major renovations required for the modernization of research laboratories for future decades. Current research space does not comply with new Federal and State health, safety, and ADA standards; is poorly configured, and the mechanical and electrical systems have reached their service potential and are in need of replacement or refurbishment. To accomplish these goals, funding from the Academic Research Infrastructure Program, combined with resources from the State of California, will be used to carry out a comprehensive renovation of Bonner Hall. Specifically, NSF funds will be used to modernize and upgrade all research laboratories and support facilities in the building to enhance functional interactions between laboratories designated for Developmental Biology, Mammalian Development and Differentiation, and Mammalian and Eukaryotic Molecular Genetics. Antiquated autoclaves, fume hoods, dishwashers, and other items of major fixed equipment, will be replaced and located in common rooms accessible to all users. The vivarium will be upgraded to conform with regulatory mandates for animal care, and laboratories currently dispersed in other buildings will be consolidated, creating an interactive environment for faculty and students. Modern facilities are needed for scientists who use tools of molecular genetics to unravel the more complex processes of life, such as embryogenesis and differentiation. Approximately 14 faculty members, 110 post-doctoral students, 80 graduate students, 60 undergraduate students and 35 staff researchers will benefit from the renovation. The project will also serve as a recruitment tool in the department's effort to increase the number of faculty representing minority groups.

DESCRIPTION: (Adapted from applicant's Abstract) Neuronal nicotinic acetylcholine receptors (AChRs) will be studied using stably transfected QT-6 quail cell lines to express epitope-tagged chick neuronal AChR gene constructs under inducible promoters. a7 AChRs will be given special emphasis because of their unusual properties (they function as homomers, have high calcium permeability and are located extrasynaptically in ciliary ganglia). Also to be studied are combinations of a3, b4, a5 + b2 subunits (because AChRs of these types are found postsynaptically in ciliary ganglia). The cell lines will be used to analyze subunit interactions, assembly pathways, and functional properties. Surface AChRs will be quantitated using toxins and mAbs to the epitope tags. Function will be assayed by whole cell patch clamp recording. Assembly intermediates will be identified by pulse labeling and immune precipitation. Subunit composition will be analyzed by immunoprecipitation and immunoblot analysis using mAbs to subunits and tags. Distribution within the cells will be evaluated by confocal microscopy. Neuronal cell lines which express a7 will also be used for comparison. These studies should reveal important aspects of the cell and molecular biology of neuronal nicotinic receptors and are relevant to the role of nicotinic AChRs in nicotine addiction.

3-D analysis; 3-dimensional analysis; 3D analysis; Area; Au element; Binding; Binding (Molecular Function); Blood Coagulation Factor IV; Bungarotoxins; CRISP; Ca++ element; Calcium; Cations; Cell Body; Cell surface; Cells; Coagulation Factor IV; Computer Retrieval of Information on Scientific Projects Database; Dissociation; Docking; Electron Microscopy; Elements; Factor IV; Fingers; Funding; Ganglia; Ganglion Cysts; Ganglionic Cysts; Ganglions; Gated Ion Channel; Generalized Growth; Gold; Grant; Growth; In Situ; Institution; Investigators; Journals; Label; Ligands; Magazine; Mediating; Membrane; Membrane Proteins; Membrane-Associated Proteins; Microscope; Molecular Interaction; Molecular Neurobiology; Morphology; Myxoid cyst; NIH; NRVS-SYS; National Institutes of Health; National Institutes of Health (U.S.); Nerve Cells; Nerve Unit; Nervous System; Nervous system structure; Neural Cell; Neural Ganglion; Neurobiology, Molecular; Neurocyte; Neurologic Body System; Neurologic Organ System; Neurons; Neurosciences; Nicotinic Acetylcholine Receptors; Nicotinic Receptors; Permeability; Play; Proteins; Publishing; Receptor Protein; Relative; Relative (related person); Research; Research Personnel; Research Resources; Researchers; Resolution; Resources; Role; Site; Snake Venoms; Source; Speed; Speed (motion); Spinal Column; Spine; Structure of ciliary ganglion; Surface; Surface Proteins; Synapses; Synaptic; Synaptic Vesicles; Three-dimensional analysis; Tissue Growth; Transmission; United States National Institutes of Health; V (voltage); Vertebral column; Work; backbone; base; cell body (neuron); ciliary ganglion; density; electron tomography; gene product; in vivo; membrane structure; neural cell body; neuronal; neuronal cell body; ontogeny; particle; postsynaptic; presynaptic; protein function; receptor; reconstruction; social role; soma; synapse formation; synaptogenesis; transmission process; voltage

The distribution of voltage sensitive sodium channels on axons in the dorsal and ventral spinal roots of the dystrophic mouse 129/ReJ-Lama2dy was determined using immunocytochemistry. In these nerves there are regions in which Schwann cells fail to proliferate and myelinate axons in a normal fashion, leaving bundles of closely packed large diameter amyelinated axons. We have identified discrete and focal concentrations of sodium channel immunoreactivity on these axons by confocal immunofluorescence, immunoelectron microscopy and Intermediate Voltage Electron Microscopy (IVEM) using a peptide-derived polyclonal antibody. In addition, simultaneous labeling with an antibody recognizing neuronal-specific ankyrinG revealed a distinct colocalization with the sodium channels on both normal and amyelinated axons. The presence of patches of sodium channels along with their anchoring protein on amyelinated axons in the absence of intervening Schwann cells demonstrates that axons can independently form and maintain these initial aggregations. This confirms that direct contact between Schwann cell and axon is not required for the formation of sodium channel patches of nodal dimensions and density. Furthermore, this strongly suggests that local transfer of sodium channels from Schwann cells to axons is not required for this process. This work was published in the Journal of Neuroscience (Deerinck et al., J. Neurosci., 17: 5080-5088, 1997).

DESCRIPTION (From applicant's abstract): Nicotinic acetylcholine receptors containing the alpha7 gene product (alpha7-nACHRs) are among the most abundant in the nervous system and have been implicated in a wide variety of higher brain functions and neurodegenerative diseases. Because of their high calcium permeability and diverse locations, the receptors can influence many cellular events. The present renewal application is based on three new discoveries: the receptors can be concentrated on somatic spines by cytoskeletal interactions for synaptic signaling; receptor function may be constrained substantially by negative regulation in vivo; and the receptors can influence gene expression in novel ways. These findings shape the four specific aims proposed. (1) Identify components that tether alpha7-nAChRs at synaptic sites. (2) Analyze mechanisms controlling the responsiveness of alpha7-nAChRs. (3) Assess the significance of alpha7-nAChR signaling for gene expression. (4) Test CNS models of postsynaptic alpha7-nAChRs. The initial work will be carried out with chick ciliary ganglion neurons because they provide the richest known source of the receptors. The neurons will be examined both in vivo and in cell culture to identify cell-cell interactions and receptor-associated molecules that govern somatic spine formation and alpha7-nAChR clustering. Exogenous components that dramatically increase the whole-cell alpha7-nAChR response will be examined for molecular mechanism and for in vivo relevance. The conditions and mechanisms enabling alpha7-nAChR receptors to influence gene expression will also be examined, and the gene families affected will be identified in order to test hypotheses about the physiological relevance of the effect. CNS neurons enriched in alpha7-nAChRs will then be used to determine which of the regulatory principles governing ciliary ganglion receptors can be extended broadly across systems having different physiological assignments. The experimental approaches will utilize fluorescence imaging, 3-D tomographic EM reconstruction, and whole-cell patch clamp recording in situ and in cell culture. These will be combined with biochemical and molecular biological approaches to identify and examine molecules influencing receptor function and location, and to determine which gene families are most affected by the receptors. The information obtained will indicate how a major nicotinic receptor in brain is controlled and how it, in turn, exerts long-term control through gene regulation. The biomedical relevance is substantial because of the enormous health-related consequences of tobacco usage and nicotine addiction and because of growing evidence that alpha7-nAChRs in particular are involved in neurodegenerative diseases such as Alzheimer's with a devastating toll on the public.

Alzheimer's Disease Drug Discovery. Alzheimer's disease is a physiologically devastating condition that is threatening to reach epidemic proportions as the population ages. Cholinergic deficit is a widely recognized aspect of Alzheimer's disease, and is now thought to include nicotinic signaling as a critical component. One of the most interesting nicotinic receptors is the subtype composed of alpha7 subunits because it is widely expressed in the nervous system and has a high relative permeability to calcium which enables it to control diverse cellular functions. Recently it has been shown that alpha7-containing receptors bind the beta-amyloid peptide (amino acids 1-42) in the pM- nM range. The mammalian hippocampus is an interesting system for studying such interactions because alpha7-containing receptors are relatively abundant there, and the hippocampus is centrally involved in memory formation which is a major target of Alzheimer's disease. In preliminary studies we have found that the beta-amyloid peptide in the nM range reversibly inhibits alpha7-containing nicotinic receptors on hippocampal neurons in culture. Surprisingly, certain peptides applied exogenously can quickly and reversibly potentiate the alpha7- containing receptor response by an order of magnitude. This raises the real possibility that drugs capable of accessing the same site may be able to compensate for beta-amyloid inhibition of the receptors. This pilot proposal will examine the interactive of beta-amyloid peptide with hippocampal alpha7-containing receptors both functionally and physically. In addition, it will test the hypothesis that exogenous application of appropriate regulatory molecules will overcome the inhibition mediated by beta-amyloid peptide. The approach will use whole-cell patch clamp recording (usually in perforated patch mode) from the neurons while applying test compounds in desired sequences from a multi-bore rapid applicator. Competition binding experiments with radiolabeled probes will also be used to assess direct interactions. Functional characterization will include modulation of spontaneous synaptic events in the cultures by alpha7-containing receptors, and determining the impact of beta-amyloid peptide on the modulation in the presence of absence of the candidate potentiators. If this pilot study proves successful (as the preliminary experiments encourage us to think it will), the results will provide the basis for a subsequent full-scale follow-up to analyze beta-amyloid effects on CNS alpha7-containing nicotinic receptors, to analyze Alzheimer's brain tissue for specific involvement of alpha7-receptors in the disease, and to evaluate compounds capable of crossing the blood-brain barrier and potentiating the receptors in situ as a possible therapeutic strategy.

DESCRIPTION (provided by applicant): This application will examine the formation of nicotinic synapses on neurons. Despite much information about the vertebrate neuromuscular junction and a variety of synapses in the CNS, almost nothing is known about postsynaptic components at nicotinic synapses on neurons, other than the identify of the nicotinic acetylcholine receptor (nAChR) itself. Given the prevalence of nicotinic signaling and its implication in a variety of neural functions and disorders, it is becoming increasingly important to understand the assembly and regulation of the molecular structures involved. Nicotinic receptors containing alpha7 subunits (a7-nAChRs) are among the most abundant in the nervous system and regulate calcium-dependent events, due in part to their having a high relative calcium permeability. Recent studies on ciliary ganglion neurons show the receptors are concentrated on somatic spines where they utilize calcium influx to control events ranging from immediate receptor modulation to long-term transcriptional control. Preliminary results suggest that both cadherin-related neuronal receptors (CNRs) and PDZ-containing proteins are associated with nicotinic synapses on the neurons and may play important organizational and regulatory roles. The receptors also form clusters on hippocampal neurons in culture but are often associated with presumptive non-nicotimc synapses where they are regulated by neural activity. These findings provide the basis for the following aims: (1) Complete 3D tomographic EM analysis of a complete nicotinic calyx synapse to elucidate structure-function relationships, (2) Identify molecular components driving nicotinic synapse formation on neurons, beginning with CNRs and PDZ-containing proteins, (3) Examine postsynaptic determinants of a7-nAChR function that control their contributions to synaptic signaling, and (4) Use rat hippocampal cultures to examine the regulation and significance of synaptic a7-nAChRs on mammalian CNS neurons. The experimental approaches include confocal and EM imaging, gene cloning and manipulation of dominant negative constructs, immunocytochemistry and immunopurification, and patch clamp recording in culture and in situ. The expected results should identify postsynaptic components that link nAChRs to regulatory and signal transduction elements and tether them to the cytoskeleton. This information will provide insight into the organization of nicotinic synapses and suggest mechanisms controlling their development and function. The biomedical relevance stems in part from the role of nicotinic signaling in fundamental brain function such as learning and memory, and in related disorders such as Alzheimers disease and nicotine addiction.

DESCRIPTION (provided by applicant): Nicotinic signaling is widespread in the nervous system and influences numerous behaviors and neuropathologies. The signaling depends on nicotinic receptors which often promote calcium influx and regulate calcium-dependent events. The consequence of signaling, therefore, can depend critically both on receptor location and on the identity of associated intracellular components. Recently it has been shown that members of the PSD-95/SAP90 family form a postsynaptic PDZ-scaffold associated with nicotinic receptors on neurons. The scaffold not only helps mediates downstream signaling but also plays an important but unknown role in supporting synaptic input to the neuron. Preliminary results suggest that postsynaptic neuroligin, EphB2 receptors, and rapid activity-driven SNARE-dependent receptor trafficking also converge in the postsynaptic neuron to regulate nicotinic signaling. Four specific aims are proposed to pursue these findings: (1) Determine how postsynaptic PDZ-scaffolds influence synaptic input and identify possible intermediate components exerting the effect. (2) Test the hypothesis: that postsynaptic neuroligin supports nicotinic input independently from PDZ-scaffolds and determine how it interacts with nicotinic receptors. (3) Examine the mechanisms promoting rapid trafficking of nicotinic receptors on neurons and assess their physiological significance for synaptic signaling. (4) Test the hypothesis that EphB2 receptors enhance nicotinic effects and examine the interdependence of convergent pathways controlling nicotinic signaling in neurons. Fluorescence imaging will be used to visualize synaptic components; transfections will be used to manipulate interacting partners; electrophysiological analysis will be used to study functional consequences; and biochemical and molecular biological techniques will be used to probe interactions. Experiments will focus mainly on chick ciliary ganglion neurons but will also employ transfected cell lines to define molecular interactions, and rat hippocampal neurons to assess the generality of the findings. These results will provide new insight into regulatory mechanisms that shape nicotinic signaling, important because the signaling is pervasive and has profound biomedical consequences.

Nicotinic acetylcholine receptors (nAChRs) are widely expressed in the nervous system and have been implicated in a variety of behaviors and neuropathologies;In most cases the receptors reach their highest levels during late embryogenesis and early postnatal life. Almost nothing is known, however, about the developmental roles of neuronal nAChRs, other than that they are cation-selective and can regulate calcium- dependent events. We have preliminary evidence that during development nAChRs utilize signal transduction to regulate synaptic capabilities, including that of adjacent non-cholinergic pathways, in both the CNS and autonomic nervous systems. This proposal will test specific hypotheses about the roles of nicotinic input in shaping the development of nicotinic and GABAergic signaling, and will examine candidate molecules that may facilitate or transduce nicotinic effects. In Aim I the chick ciliary ganglion is used to test hypotheses about the significance of newly discovered GABAergic pathways in this cholinergic ganglion, the role of nicotinic input in controlling GABAergic maturation from excitatory to inhibitory mode, and the consequences this has for ganglionic throughput. In Aims II and III, the mammalian hippocampus is used to test hypotheses about the role of nicotinic signaling in regulating filopodia fate, induction of dendritic spines, maturation of GABAergic signaling from excitatory to inhibitory, and acute modulation of GABA responses. Long-lasting nicotine sensitization is also examined. In Aims IV and V three candidate proteins that appear to specifically interact with neuronal nAChRs are examined for their roles in determining the number, location, function, and downstream signaling capabilities of the receptors. Each of these aims is based on preliminary data supporting the central hypotheses being tested. The experimental approaches include electrophysiology, imaging, molecular biology, and biochemistry, and are carried out on neurons in cell culture and slice preparations from wildtype and mutant sources. Together these approaches and systems should provide mechanistic insight into novel roles of nicotinic signaling in the developing nervous system. The findings should also have biomedical relevance in revealing the consequences of nicotine exposure during these formative periods.

Nicotinic cholinergic signaling uses the transmitter acetylcholine to activate ligand-gated ion channels that are cation-selective in mammals. This form of signaling is widespread in the nervous system, reaches peak levels during early postnatal life, and continues throughout adulthood. It contributes to a variety of behaviors including arousal and cognition, participates in a number of neurodegenerative disorders including Alzheimer's and Parkinson's diseases, and is responsible for nicotine addiction. Despite intensive effort, little is understood about the role of nicotinic signaling during development when it drives spontaneous waves of excitation across the nervous system, and little is understood about the nicotinic mechanisms that subsequently exert global effects across networks in the adult brain. This proposal tests two novel hypotheses fundamental to these issues. The first is that nicotinic signaling during development promotes the formation of glutamatergic synapses both on early postnatal and adultborn neurons (Aims I & II). Since glutamatergic pathways provide the principal form of excitation in brain, this effect of nicotinic input is likely to have lasting consequences for nervous system function. The second hypothesis is that nicotinic activity in the adult brain can acutely and reversibly alter GABAergic signaling such that it stops being inhibitory and transiently becomes excitatory (Aim III). This could exert far-reaching effects across networks radically altering output. Preliminary studies performed on the hippocampus strongly support these ideas. The two major nicotinic acetylcholine receptors, homopentameric ¿7-nAChRs and heteropentameric ¿2-containing nAChRs, appear to complement each other in promoting glutamate synapse formation. One appears to act in cell-autonomous fashion to drive postsynaptic spine formation while the other may act indirectly to recruit components required for synaptic function. Preliminary results also support the second hypothesis: low levels of nicotine experienced by tobacco users may be sufficient to transiently invert the chloride gradient in adult neurons, thereby rendering GABA temporarily depolarizing. This could dramatically change the excitability of networks housing those neurons. The hypotheses will be tested by pharmacological and genetic manipulation, including loss-of-function and rescue experiments, performed on hippocampal slices and in vivo. The underlying molecular mechanisms will be analyzed and their consequences evaluated for system function. Imaging and electrophysiological approaches will be combined in this analysis. The experiments proposed here test pivotal ideas about the purpose of nicotinic cholinergic signaling in the nervous system. The results are likely to change how we think about fundamental processes guiding development and regulation of function in neural networks. The vulnerability of these processes to exploitation by tobacco-derived nicotine gives this work compelling health-related significance.

Glutamatergic pathways comprise the principal excitatory networks in the central nervous system, employing a combination of synapses on dendritic spines and shafts to position neurons in circuits. How synaptogenesis can be coordinated across neuronal populations during development to form these networks remains poorly understood. Our preliminary results suggest a profound and novel role for endogenous nicotinic signaling in this process; it appears to be a major force driving development of glutamatergic pathways and determining the kinds of connections formed. Nicotinic activity results from the transmitter acetylcholine (ACh) activating ionotropic nicotinic ACh receptors (nAChRs). The receptors appear early in postnatal life and initially mediate spontaneous waves of excitation in many brain regions. The two most abundant nAChR subtypes are ¿7- containing homopentamers (¿7-nAChRs) and ¿2-containing heteropentamers (¿2*-nAChRs). Our preliminary results with brain tissue from mice lacking ¿7-nAChRs (constitutive knockouts) using multiple methods suggests that the receptors are required for neurons to receive proper numbers of functional glutamatergic synapses during early postnatal life. Preliminary results with mice lacking ¿2*-nAChRs indicates the receptors provide a complementary role, increasing the number of dendritic spines available to receive glutamatergic synapses. In this proposal we combine genetic manipulation, imaging, patch-clamp recording, network labeling, and ultrastructural analysis to examine how nicotinic activity influences the synaptic composition of networks in vivo. Three Specific Aims are proposed. The first tests the hypothesis that endogenous nicotinic cholinergic signaling determines the number, location, and possibly source of glutamatergic synapses on neurons in early postnatal brain and that the effects persist into the adult, determining the ratio of glutamatergic/GABAergic input a neuron receives. The second tests the hypothesis that early exposure to nicotine in vivo, at levels encountered by smokers, disrupts normal development, produces excessive glutamatergic contacts, and imposes long-lasting changes in neural circuits. The third tests the hypothesis that nicotinic control of spine formation is rapid and local, whereas nicotinic control of synapse formation involves microRNAs for broad coordination of gene expression. The experiments employ new methods, including mapping techniques in vivo to distinguish network changes and physiological methods to assess functional consequences. The findings are likely to have major import both for prevailing views about nicotinic cholinergic function during development and for understanding mechanisms guiding network construction. The results will have direct biomedical relevance in revealing mechanisms by which early nicotine exposure is likely to produce long-lasting behavioral impairments that continue in adults. Targets may be suggested for therapeutic intervention to protect against or compensate for prior nicotine exposure. These results will represent a major step forward in understanding nervous system development and identifying key vulnerabilities.

DESCRIPTION (provided by applicant): Glutamatergic synapses comprise the major excitatory pathways in brain. A key feature determining the strength of a glutamatergic synapse is the number of postsynaptic AMPA receptors available for activation. We have preliminary evidence that nicotinic stimulation of astrocytes causes them to release a component that specifically recruits AMPA receptors to postsynaptic sites on neurons, rendering these previously silent synapses functional. This is completely unexpected and suggests a new and important mechanism for shaping excitatory circuits. Astrocytes are pervasive and abundant when glutamatergic synapses are first forming during development, and have significant numbers of ?7-containing nicotinic acetylcholine receptors (?7-nAChRs). Spontaneous nicotinic cholinergic activity mediated in part through ?7-nAChRs is also widespread early in development. Our preliminary evidence suggests that it is these ?7-nAChRs that are responsible for the astrocyte effect on AMPA receptors. In view of the early appearance and widespread occurrence of astrocytes and ?7-nAChR signaling, such a mechanism could have profound consequences for shaping network formation and functional output. The first Specific Aim will test in cell culture the synaptogenic effects of components released uniquely by nicotinic stimulation of astrocyte ?7-nAChRs. Pre- and postsynaptic components at glutamatergic synapses will be examined, synaptic function assessed, and a screen of candidate components initiated. The second Specific Aim will test the hypothesis in vivo, using viral constructs and a GFAP-Cre system in mutant mice to determine if astrocyte ?7-nAChRs play a critical role in recruiting postsynaptic AMPA receptors on neurons in the hippocampus. Subsequent experiments will test whether astrocyte ?7-nAChRs also contribute to synaptic plasticity, focusing on locations where previous studies have shown that long-term potentiation results from increased numbers of AMPA receptors. In addition to providing new insight into fundamental processes in the brain, these studies will have serious biomedical implications. Finding that nicotinic signaling influences such basic features as the functionality of glutamatergic synapses may put the system at high risk during development if exposed early on to nicotine, such as through secondhand smoke or maternal milk, either prematurely stimulating or desensitizing this regulatory input. Further, the results will have immediate relevance for pharmaceutical companies globally targeting ?7-nAChRs in drug design without realizing that astrocyte ?7-nAChRs may have this unique and critical role. This R21 project will test the central hypotheses and lay the groundwork for future projects understanding the role of astrocytes and nicotinic signaling in regulating nervous system form and function.

DESCRIPTION (provided by applicant): The developing nervous system undergoes a major transition in early postnatal life when it shifts from a period of exuberant synapse formation to a subsequent period of synaptic pruning and network consolidation. Synaptic numbers approach a maximum during this time, the rules governing synaptic plasticity change, AMPA and NMDA receptor compositions are altered, and GABAergic signaling shifts from being depolarizing/excitatory to hyperpolarizing/inhibitory, in addition to other changes. Mechanisms driving this profound transition are poorly understood at best. Interesting candidates for doing so are microRNAs (miRs) because they can target many different mRNAs at the same time for blockade or destruction, thereby producing a regulatory hub to coordinate complex changes across large systems. Our preliminary evidence indicates that miR-101 is an attractive candidate for executing this transition. It is abundant, increases substantially at the relevant time, and remains high in the adult. Using antagonists to block miR-101 function in vivo reveals significant aberrations: substantial increases are seen in spontaneous synchronized activity across neuronal populations and is accompanied by increases in synaptic number and excitatory synaptic input to neurons. Blocking miR-101 also appears to delay maturation of GABAergic signaling, enabling it to be depolarizing at later times. Importantly, our preliminary results using target site blockers to protect specific mRNAs suggest that miR-101 achieves its effects by acting on multiple targets. One is likely to be NKCC1, the chloride transporter responsible for GABA being depolarizing. But other miR-101 targets appear to be important as well, suggesting that terminating the depolarizing phase of GABAergic signaling is not by itself sufficient to account for all the major changes comprising the developmental transition. We will test the role of miR-101 in mediating the transition in nervous system development by using antagonists, sponges, and target site blockers to prevent its action in vivo while assessing the consequences for synaptic activity and network function. Calcium fluors in acute slices will report the frequency and extent of spontaneous coordinated activity across neuronal populations, as well as total activity and numbers of participating neurons. Patch-clamp recording will reveal type and amount of synaptic activity while immunostaining will quantify synapses. By selecting individual miR-101 targets for protection, it will be possible to dissect th contributions of different pathways to the transition and to evaluate the consequences of defective regulation. This will provide new insight into mechanisms driving the transition, reveal the role of miR-101 in particular, and likely have biomedical relevance by revealing the vulnerability of the system to individual regulatory pathways being compromised, producing, for example, greater propensity for epileptic seizure-like events.

? DESCRIPTION (provided by applicant): Neuronal circuits in the brainstem control life-sustaining functions, in addition to driving and gating active sensation through taste, smell, and touch. We propose to exploit the advent of molecular and genetic tools to undertake cell lineage marking, cell phenotyping, molecular connectomics, and methods from machine learning and image processing to construct an integrated anatomical and functional atlas of the brainstem. This will enable us to generate anatomical wiring diagrams for the brainstem circuits that control or facial actions. There are three phases to this work. (1) Reveal the identity and organization of brainstem nuclei. Motivated by striking similarities between the developmental plan for the spinal cord and brainstem, we will embrace and extend these efforts to interrogate the molecular composition of neurons that define individual nuclei with sensorimotor circuits in the murine brainstem. (2) Reveal brainstem neuronal circuits and their interactions. We will utilize Tran synaptic viral labeling to delimit pathways from specific muscles that are innervated by facial, trigeminal, hypoglossal, and laryngeal motor nuclei. This will reveal hitherto unknown brainstem circuits, including sites of modulation by higher brain areas. (3) Control the behavior of identified feedback circuits. We will manipulate specific populations of brainstem neurons using a battery of genetic tools to delineate or facial motor actions and motor synergies. The results from the above efforts will be a quantitative map of the functional organization of neurons in the brainstem that enable studies on computations that underlie or facial behavior. An understanding of these fundamental behaviors bears directly on the more general issue of how nervous systems deal with computations that can be performed autonomously, yet must interact synergistically. Thus our proposed program on brainstem circuitry and dynamics will yield general lessons about the nature of neuronal computation. The work performed under this proposal will serve as the basis for a larger national effort in brainstem neuronal computation.

? DESCRIPTION (provided by applicant): Early nicotine exposure during brain development produces long-lasting behavioral changes that are detrimental in multiple ways. These include greater propensities for nicotine addiction, attention deficit hyperactivity disorder, anxiety, and depression. The mechanisms remain unclear. We have preliminary evidence indicating that early exposure of mouse pups to nicotine from the lactating mother during nursing (postnatal day 2-16) produces long-lasting increases in the number of glutamatergic synapses and increases in the ratio of excitatory-to-inhibitory synaptic input neurons receive. The preliminary results also indicate that even after a long period of nicotine abstention, the mice as adults have abnormally large numbers of neurons that display high levels of activity when re-challenged briefly with nicotine. This can be seen either in acute slices or in alive animals with in vivo imaging. Such changes at the cellular level are new and likely to contribute importantly to the long-lasting behavioral changes reported. To examine the underlying mechanisms and evaluate their consequences, we propose the following. We will use immunostaining and patch-clamp recording from neurons in acute slices to determine the extent of increases in the glutamatergic input they receive after nicotinic exposure from the mother via nursing or in utero. We will determine whether the changes extend into adulthood long after nicotine cessation, and whether such animals are more vulnerable to network changes when challenged with subsequent nicotine than are naïve animals. Further, we will determine whether subpopulations of neurons under these conditions can be identified by expression of the immediate early gene c-fos, characteristic of high activity, and we will also use in vivo imaging of awake mice to examine the properties of hyper-active cells. Neuronal populations to be examined include pyramidal neurons in the hippocampal CA1 because of their roles in memory formation and dopaminergic neurons in the ventral tegmental area because of their participation in reward pathways. These studies will identify mechanisms and pathways contributing to the long-lasting effects of early nicotine exposure. They will provide new insight into mechanisms guiding important aspects of circuit formation and brain development. Importantly, they will also have significant biomedical relevance because of current medical policy recommending nicotine replacement therapy for pregnant women who smoke. That procedure, together with the increasing usage of electronic cigarettes, pose serious health threats that are insufficiently understood. The results obtained here will help clarify the consequences and indicate new strategies for exploration.