Rita Balice-Gordon - US grants

This proposal describes a series of experiments which will address how neural activity shapes patterns of synaptic connections in the developing nervous system. Once established, synaptic connections undergo a prolonged editing process which results in some functional connections becoming permanently deleted while others are maintained. Work over the last 20 years in different parts of the nervous system has shown that this synapse elimination involves competitive interactions among different inputs innervating the same target cell and is modulated by neural activity. Following this period, ongoing maintenance of synaptic connections and their use-dependent modulation continue to occur over a lifetime. How axons form synapses within a target, begin to function and become edited to give functionally relevant patterns of connectivity are poorly understood. In large part, this is because dynamic events such as these are difficult to study in vivo and have proved to be difficult to recapitulate in vitro. Neuromuscular junctions are well suited to studying activity dependent modulation of synapse formation and elimination in vivo, because they are simply organized, easily observed and manipulated, and there exists a wealth of information about their structure, function and molecular composition. We will use in vivo video microscopy and physiological analysis to study early structural and functional events during synapse formation and elimination at mouse neuromuscular junctions. We will then alter the molecular environment between motor neurons and muscle fibers in a spatially and temporally selective manner using viral mediated gene transfer. We will study the role of neural activity in modulating synaptic competition in in vitro preparations of spinal cord, nerve and muscle and with in vivo pharmacological manipulations. of particular interest is determining whether the disappearance of electrical coupling among motor neurons innervating the same muscle fiber triggers the onset of synaptic competition. These three sets of experiments are aimed at elucidating the activity-dependent and -independent mechanisms that result in functional patterns of synaptic connections throughout the developing nervous system.

Balice-Gordon
IBN-9982233

A fundamental question in neuroscience is how connections are made and maintained among neurons and their targets. A number of studies over the last 25 years have shown that neural wiring is exquisitely sensitive to experience, which is translated into neural activity. Thus changes in neural activity can have a profound impact on neural connectivity. Despite this appreciation, the underlying mechanisms are poorly understood. We use the synaptic connection between motor neurons and muscle fibers, called neuromuscular junctions, as a model system to understand how neural activity changes neural connectivity. We propose to study how the relative firing patterns of motor neurons change during postnatal development, and whether such change might modulate changes in synaptic connections at developing neuromuscular junctions. During late embryonic and early postnatal life, skeletal muscle fibers undergo an activity-dependent, competitive transition from multiple to single innervation. In developing muscle and brain, competition among motor units is modulated by their relative patterns of activity. Temporally correlated patterns of motor unit activity might be present before competition begins, while asynchronous activity may lead to the loss of the least active inputs. While this hypothesis is supported by many experimental manipulations, no studies have determined the relative activity patterns of motor neurons that impinges on neuromuscular synapses in vivo. These experiments will also further our understanding of how neural activity shapes patterns of synaptic connectivity, not only during development, but throughout life.

DESCRIPTION (provided by applicant): This revised proposal is aimed at determining the role of neurotrophins that signal through Trk receptors in modulating synapse structure and function in the developing and adult nervous system. The mouse neuromuscular junction will be used as a model system. The cell types that comprise neuromuscular junctions, the perisynaptic Schwann cells, presynaptic motor neuron terminals, and postsynaptic muscle fibers, each express a complement of neurotrophins and Trks, suggesting that neurotrophin signaling at this synapse is multi‑directional and involves all three cell types. Similarly, CNS neurons express several neurotrophins and Trks, but the relative roles of each signaling pathway in synaptic maturation and maintenance are unclear. Recent work from our lab showed that TrkB isoforms (which bind the neurotrophins BDNF and NT4/5) is expressed primarily postsynaptically, in the muscle fiber membrane in and around acetylcholine receptor (AChR) rich regions, while TrkC isoforms are localized to perisynaptic Schwann cells and TrkA is not localized to neuromuscular junctions. Down-regulation of TrkB signaling in muscle fibers, via adenovirus-mediated over-expression of a truncated, non-signaling form of TrkB (TrkB.tl), induced the dismantling of postsynaptic AChR rich regions. These observations lead to the hypothesis that exchange of ligands that signal through TrkB or TrkC receptors play different roles in synaptic maturation and maintenance. To test this hypothesis, we will use adenovirus‑mediated manipulation of neurotrophin and Trk expression in different cell types at neuromuscular junctions in vivo. The effect of these manipulations on neuromuscular junction structure and function will be analyzed with in vivo imaging, immunostaining, confocal microscopy and electrophysiological characterization of synaptic strength. We will also explore the molecular mechanisms downstream of TrkB‑mediated signaling, and how these may interact with the agrin/MuSK signaling pathway that mediates AChR clustering. The results of these experiments will provide new insights into the functional role(s) of neurotrophin and Trk‑mediated signaling at developing and adult synapses, and extend our understanding of the relative roles of antero‑ and retrograde synaptic signaling in the peripheral as well as central nervous system.

DESCRIPTION (provided by applicant): This proposal is aimed at determining the role of neurotrophins that signal through Trk receptors in modulating synapse structure and function in the developing and adult nervous system. Mouse neuromuscular synapses will be used as a model system. The cell types that comprise neuromuscular junctions, the perisynaptic Schwann cells, presynaptic motor neuron terminals, and postsynaptic muscle fibers, each express a complement of neurotrophins and Trks, suggesting that neurotrophin signaling at this synapse is multi-directional and involves all three cell types. Similarly, CNS neurons express several neurotrophins and Trks, but the relative roles of each signaling pathway in synaptic maturation and maintenance are unclear. Previous work and preliminary results from our lab showed that TrkB isoforms (which bind the neurotrophins BDNF and NT4/5) are expressed primarily postsynaptically, in the muscle fiber membrane in and around acetylcholine receptor (AChR) rich regions, while TrkC isoforms are localized to perisynaptic Schwann cells and TrkA is not localized to neuromuscular junctions. Down-regulation of TrkB signaling in muscle fibers, via adenovirus-mediated over-expression of a truncated, non-signaling form of TrkB (TrkB.t1), induced the dismantling of postsynaptic AChR rich regions. In contrast, preliminary results show that TrkC modulates the extension of processes by these cells that play important roles in axon sprouting and reinnervation. These observations lead to the hypothesis that exchange of ligands that signal through TrkB or TrkC receptors play functionally distinct roles in synaptic maturation and maintenance. While adenoviral methods are useful, and are the supported by another grant, to determine the role of these signaling molecules at synapses, it will be essential to selectively delete neurotrophin or Trk genes from one of the cell types at neuromuscular synapses. Because many of the relevant mutations in mice result in perinatal lethality, we will use cre-mediated recombination to delete neurotrophins or Trks from cells of interest in a spatially and temporally controlled fashion. The effect of neurotrophin (BDNF, NT3) or TrkB deletion on neuromuscular synaptic structure and function will be analyzed with in vivo imaging, immunostaining, confocal microscopy and electrophysiological characterization of synaptic strength. The results of these experiments will provide new insights into the functional role(s) of neurotrophin and Trk-mediated signaling at developing and adult synapses, and extend our understanding of the relative roles of antero- and retrograde synaptic signaling in the peripheral as well as central nervous system.

DESCRIPTION (provided by applicant): I propose to study the mechanisms by which astrocytes modulate inhibitory synaptogenesis in the developing central nervous system. Previous work and preliminary studies in hippocampal neurons in vitro show that astrocytes secrete proteins into the media (astrocyte conditioned media, ACM) that increase inhibitory neuron axon length, branching as well as synaptogenesis, by the criteria of increasing the number of GABAergic presynaptic terminals co-localized with postsynaptic GABAAR clusters (Elmariah et al., 2005;Hughes et al., 2005, 2006). These data lead to the hypothesis that astrocyte secreted proteins affect the formation of inhibitory circuitry during neural development. This hypothesis will be tested in three aims: (1) We will complete our studies to determine how astrocyte secreted proteins affect inhibitory axon length, branching and synaptogenesis in 2 experiments. (2) We will continue to use gel filtration and mass spectroscopy to identify astrocyte secreted proteins that increase inhibitory axon length, branching and synaptogenesis. (3) We will test candidate secreted proteins for their role in inhibitory axon length, branching and synaptogenesis. While these aims are high-risk, they are also potentially of very high impact, as very little is known about astrocyte secreted proteins that affect inhibitory neurons. Taken together, these experiments will extend our understanding of how inhibitory circuitry is formed during neural development, and may also contribute to understanding of disorders of development such as epilepsy, autism and mental retardation. PUBLIC HEALTH RELEVANCE: I propose to study the signaling mechanisms by which astrocytes, a type of glia in the brain, affect inhibitory neurons and the formation of inhibitory synapses in the developing central nervous system. The proposed experiments will contribute to understanding of disorders of human nervous system development such as epilepsy, autism and mental retardation, disorders with a significant public health impact.

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (01): Behavior, Behavioral Change, and Prevention and specific Challenge Topic 01-AA-102: Functional Roles of Neuroimmune Factors in Mediating Behavior. The focus of this proposal is the characterization of autoimmune responses to synaptic proteins that result in disorders of behavior, memory, cognition and psychosis. In 2007, we first reported a group of young women who acutely developed psychotic behavior or schizophrenia, subsequently followed by decrease of memory, catatonia, abnormal movements, and autonomic dysfunction. Using techniques that we optimized to detect antibodies to neuronal cell surface and/or synaptic proteins, we found that all patients had antibodies against the NR1 subunit of the NMDA receptor, a glutamate receptor that plays important roles in synaptic transmission and plasticity. In about 60% of patients, the immune trigger was an ovarian teratoma with ectopic nervous tissue and expressed NMDAR. Since that report, the number of patients diagnosed with this disorder has rapidly increased, and similar strategies applied to patients with other neuropsychiatric manifestations have led to the discovery of 4 novel immune responses to cell surface/synaptic autoantigens, including, among others, the GluR1/2 subunits of the AMPA receptor, and the GABA(B1) receptor. Our recently published studies have shown that patients'NMDAR or AMPAR antibodies reduce the number and synaptic localization of receptor clusters in dissociated hippocampal neurons in vitro, and that these effects are reversed upon removal of antibodies from the culture medium. These findings have led to the hypothesis that many acute encephalopathies of unknown etiology causing behavioral, personality and memory deficits are likely mediated by antibodies that affect neurotransmitter receptors at cell surface or synaptic sites. In 3 disorders for which preliminary studies show CSF antibodies to cell surface/synaptic proteins, including rapidly progressive psychosis;acute behavioral deficits, language dysfunction and mutism in children;and acute memory deficits and anterograde amnestic syndromes, we will perform 2 aims: 1) Determine the identity of the autoantigens in these 3 disorders, using modified highly sensitive methods to detect the presence of antibodies to neuronal cell surface/synaptic antigens;and 2) Determine how patients'antibodies modify the structure and function of synapses in rodent neurons in vitro and in vivo, focusing on how the density and synaptic localization of antigens is altered by patients'antibodies, and how these recover after antibodies are removed. The results of these experiments will allow us to begin to determine the range of autoantigens that lead to encephalopathies with associated behavioral manifestations in humans, and to begin to determine the underlying molecular, cellular and synaptic mechanisms in these common and devastating disorders. We propose to screen patients presenting with encephalopathies of unknown etiology that result in personality, behavior and language dysfunction for autoimmune processes. We hypothesize that patient antibodies affect neurotransmitter receptors in the neuronal membrane and at synapses, leading to changes in synaptic and circuit function that in turn lead to behavioral, personality and memory deficits. The results of the proposed experiments will provide fundamentally new insights into the molecular, cellular, synaptic and behavioral mechanisms underlying anti-glutamate receptor encephalopathies, provide new insights into memory and cognitive deficits that are hallmarks of these disorders, and potentially suggest avenues for therapeutic intervention in these common and devastating disorders of memory and cognition that have a significant public health impact. PUBLIC HEALTH RELEVANCE: We propose to screen patients presenting with encephalopathies of unknown etiology that result in personality, behavior and language dysfunction for autoimmune processes. We hypothesize that patient antibodies affect neurotransmitter receptors in the neuronal membrane and at synapses, leading to changes in synaptic and circuit function that in turn lead to behavioral, personality and memory deficits. The results of the proposed experiments will provide fundamentally new insights into the molecular, cellular, synaptic and behavioral mechanisms underlying anti-glutamate receptor encephalopathies, provide new insights into memory and cognitive deficits that are hallmarks of these disorders, and potentially suggest avenues for therapeutic intervention in these common and devastating disorders of memory and cognition that have a significant public health impact.

DESCRIPTION (provided by applicant): Synapses made by a neuron with its synaptic partners are malleable during development, and as a consequence of experience, with respect to number, strength, and functional properties such as short and long term plasticity. A well studied model system for developmental, activity-dependent plasticity is mouse neuromuscular synapses, which undergo activity-dependent plasticity in development that is a hallmark of their smaller, less accessible CNS counterparts. During late embryonic and early postnatal life, neuromuscular synapses undergo elimination, in which the synapses of one axon are pitted in competition against the synapses of other axons innervating the same muscle fiber. Several lines of evidence suggest that the most active axon will have the strongest synapses and emerge as the winner, maintaining single innervation of a muscle fiber, while other, less active axons will wither, lose synaptic strength, and become eliminated. However, while the structural progression of events during synapse elimination is known, and some aspects of the functional progression are known, how the two are interrelated over time is entirely unknown. Furthermore, no previous studies have directly linked temporal information about activity patterns/stimulation to changes in synaptic strength to changes in synaptic area, or have triggered synapse weakening - or synapse elimination - with differential activity at neuromuscular junctions in situ. Here we propose to test the hypothesis that at dually innervated neuromuscular junctions, the activity of one input heterosynaptically weakens the other input, preceding synapse loss, axon atrophy and input withdrawal. To test this hypothesis, we will use a line of transgenic mice in which the mouse Thy1.2 promoter drives expression of Channelrhodopsin::YFP in all sternomastoid muscle motor axons and their nerve terminals (Thy1-ChR2::YFP100). Preliminary studies in nerve- muscle preparations from neonatal and adult mice show that postsynaptic muscle fiber action potentials can be elicited for many hours by brief pulses of 488 nm laser light focused onto ChR2::YFP+ motor axons or their terminals delivered from 1 to 100 Hz. When these mice are crossed to mice that express CFP in ~50% of nerve terminals (Thy1-CFP50%), competing inputs can be spatially discriminated and differentially stimulated with light. We propose to use these mice to: 1) to determine the temporal parameters and mechanism by which stimulation of one axon causes heterosynaptic weakening of the synapses of the unstimulated axon; and 2) determine how heterosynaptic weakening of one input results in synapse loss, axon atrophy and input withdrawal. These studies will establish, for the first time, important spatial and temporal aspects of the mechanism by which activity leads to changes in synaptic strength, culminating in synapse elimination that permanently alters neural circuitry. PUBLIC HEALTH RELEVANCE: The proposed studies will provide important, new information on the activity-dependent rules that govern the fate of developing synapses. Neuromuscular synapses in developing mice, whose maintenance is modulated by neural activity, will be used as a model system. These studies will establish important and previously unknown spatial and temporal features of the mechanism by which activity leads to heterosynaptic changes in synaptic strength, culminating in synapse elimination that permanently alters neural circuitry. This information is important when considering therapeutic interventions for spinal cord injury and diseases of the neuromuscular axis that compromise synaptic maintenance and culminate in motor neuron inactivity and/or death.