Susan Birren - US grants

9514552 Birren The mammalian nervous system is made up of an astonishingly large number different nerve cells (neurons) which interact to permit thought, movement and the control of bodily functions. The generation of this enormous cellular diversity is one of the central issues of developmental neurobiology. We know that the many different classes of neurons arise from a relatively small number of undifferentiated precursor cells in the mammalian embryo. The goal of this project is to understand how these precursor cells respond to signals in their local embryonic environment to develop into the correct type of neuron for a given area of the embryo. The peripheral nervous system provides an excellent system for this study. Evidence from other laboratories has suggested that the enteric neurons that control the function of the mammalian gut are very closely related to the sympathetic neurons that control involuntary functions such as heart rate. In fact, it has been suggested that these two neuronal lineages arise from a common embryonic precursor cell. We plan to directly test the idea that the precursor cells that normally develop into sympathetic neurons have the capacity to develop into enteric neurons when provided with the appropriate embryonic signals. Likewise, we will investigate whether enteric precursor cells can develop into sympathetic neurons. We will also test the idea that as these cells respond to signals and follow one of these differentiation pathways that they lose the ability to respond to signals from the other lineage. This will permit us to define the developmental time that multipotent precursor cells become restricted to specific cell lineages. Finally, we will seek to understand the nature of the signals that direct the precursor down one or the other neuronal lineage. We will study the distribution of neurotrophin receptors in the developing sympathetic and enteric nervous system. These receptors comprise a family of proteins that play a role in the de velopment of many different types of neurons. We will test the possibility that one of the members of this receptor family is directly responsible for lineage decisions in the sympathetic nervous system. We will do this by introducing the expression of this receptor into enteric and sympathetic precursor cells at early developmental stages and asking if we can observe alterations in the type of neurons developing from the precursors. These experiments will provide important information on how the early embryo controls the development of different cell types from uncommitted precursor cells. Normal development of the mammalian embryo requires that many such lineage decisions be carried out in a very precise manner. Our studies will shed light on the mechanisms that control these developmental patterns.

The development of appropriate neuronal circuitry is critical for normal function of the nervous system. Defects in the formation or modulation of synaptic connections may contribute to the etiology of a number of developmental and nervous system disorders. Yet little is known about the molecules that regulate synapse formation and function. In the peripheral nervous system, nerve growth factor (NGF) has been implicated in the innervation of target tissues by sympathetic neurons. Recent studies in this laboratory have indicated that NGF also influences the formation and strength of synapses between sympathetic neurons and cardiac myocytes in vitro. This system provides a novel opportunity to investigate both developmental and modulatory roles of neurotrophins in the establishment of neuronal circuitry. The goals of this proposal are to define the cellular and molecular mechanisms underlying NGF-mediated enhancements in synapse formation and function. Synaptic connections that form between sympathetic neurons and cardiac myocytes can be assayed physiologically by measuring evoked postsynaptic currents in myocytes, or functionally as an increase in myocyte beat rate upon stimulation of a connected neuron. The neurotransmitter dependence, neurotrophin NGF-dependent synaptic modulation will be determined in neuron/myocyte co-cultures. We will determine whether NGF acts presynaptically to alter neurotransmitter release or postsynaptically to alter the myocyte response to neurotransmitter. We will test the hypothesis that NGF acutely potentiates synaptic transmission by enhancing the release of norepinephrine from presynaptic terminals and will investigate a role for additional co-transmitters in the system. Preliminary results indicate that NGF-dependent modulation of sympathetic synapses may involve two different receptors for NGF, TrkA and p75. We will investigate whether the level of NGF-dependent synaptic modulation is regulated through two interacting signal transduction systems. Finally, we have demonstrated long-term effects of NGF on the development of sympathetic synapses. We will investigate whether NGF determines the number or strength of synapses that develop between sympathetic neurons and cardiac myocytes. These experiments will, for the first time, define the developmental and modulatory roles of NGF at the synaptic level.

ABSTRACT NOT PROVIDED

DESCRIPTION (provided by applicant): Bone morphogenetic proteins (BMPs) have been implicated as control factors in many different developmental transitions. During nervous system development BMPs regulate neural induction, formation of the neural crest, and the development of both neurons and glia. Within individual neural lineages, BMPs control progressive developmental decisions, reflecting changes in cellular responses to BMPs over time. In the peripheral nervous system BMPs increase neuronal differentiation of neural crest cells and later promote the maturation of enteric and sympathetic neurons along lineage-specific pathways. The goal of this proposal is to determine the cellular and molecular mechanisms underlying the developmental actions of BMPs during the divergence of the enteric and sympathetic nervous systems and to investigate the basis of changing BMP responsiveness. We will determine the role of BMPs in regulating developmental transitions of multipotent precursor cells, committed neuronal progenitors, and developing glial cells. We will test the hypothesis that BMP2 promotes neuronal differentiation by maintaining a pool of uncommitted precursor cells at the expense of glial differentiation and that BMP signaling contributes to the timing of neuron and glial development in the peripheral nervous system. A role for BMPs in the divergence of the enteric and sympathetic lineages and the maturation of specific neuronal subtypes will be examined and we will test the idea that BMP signaling contributes to the acquisition of functional neuronal properties in vitro and in the animal. BMP responses depend upon the pattern of transcriptional activation set up through expression of specific transcription factors. We will use DNA microarrays to define BMP-induced changes in patterns of transcription factor gene expression at different developmental stages. The integration of cellular and transcriptional analysis of BMP function and the use of a novel inducible transgenic mouse system for the regulation of BMP signaling in vivo will result in a new understanding of the regulatory circuits that contribute to the divergence and maturation of peripheral neuron lineages.

The sympathetic nervous system innervates a number of targets, including the heart, and sympathetic activity is a critical regulator of cardiac function. Sympathetic neurons form noradrenergic synapses onto heart cells resulting in excitation of myocyte function. Interestingly, these neurons also form cholinergic synapses onto themselves and are capable of releasing acetylcholine at neuron-myocyte synapses, which opposes the excitatory effects of noradrenergic transmission. The cholinergic and noradrenergic properties of these neurons are regulated by two neurotrophins, nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). NGF promotes noradrenergic transmission via activation of Trk receptors. In contrast, BDNF acts through the p75 receptor to increase activity-dependent acetylcholine release. While extensive work has been done defining the co-transmission profile of sympathetic neurons, little is known about how co-transmission is established in the context of multiple targets or how selective availability of neurotrophic factors regulates the development of synaptic sites and the release of neurotransmitters in a target-specific manner. We will use electrophysiological and imaging approaches to examine the idea that the development of neurotransmitter properties of sympathetic neurons is locally regulated by the expression of neurotrophins at different targets and that individual neurons can maintain multiple release profiles at different synaptic sites. We will investigate the neurotrophin receptors that regulate the development of cholinergic and noradrenergic synaptic transmission and examine the role of neurotrophin signaling in the development of cholinergic synapses in vivo. By defining the developmental mechanisms that determine the level of sympathetic drive to the heart, these studies will provide a new understanding of the neural control of cardiovascular function.

DESCRIPTION (provided by applicant): Neural control of the cardiac system depends on the opposing actions of the sympathetic and parasympathetic nervous systems. The sympathetic system is excitatory for beat rate and cardiac output, and dysregulation of sympathetic drive has been linked to a number of human disorders including hypertension and heart failure. The ability to devise new approaches for the treatment of these disorders requires an understanding of the mechanisms that control sympathetic output. These mechanisms take place at the level of sympathetic neurotransmission onto cardiac cells and the regulation of activity levels of ganglionic sympathetic neurons. While sympathetic neurons form excitatory noradrenergic synapses onto cardiac muscle cells, they are also able to synthesize and release acetylcholine, which acts as an inhibitory neurotransmitter for heart muscle and an excitatory neurotransmitter at synapses that the neurons form onto themselves. We will investigate the acute regulation of co- transmission in these neurons and determine the roles of the neurotrophic factors nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), and their receptors, in coordinating local neurotransmitter properties at neuronal and cardiac synapses. We will use electrophysiology, immunocytochemistry, and perturbation experiments to determine the role of BDNF and NGF in modulating pre- and postsynaptic properties and to examine spatially restricted effects of neurotrophins at sympathetic synapses. We will determine if neurotrophins acutely modulate sympathetic function in vivo. Finally, we will investigate neurotrophic regulation of activity levels and firing properties and will determine how integration of intrinsic and synaptic regulation defines the functional output of the cardiac system. PUBLIC HEALTH RELEVANCE: Heightened sympathetic drive to the heart is linked to a number of pathologies, including sudden cardiac death. In this project we will investigate mechanisms that acutely regulate the level of excitatory and inhibitory transmission in individual sympathetic neurons and test the hypothesis that interactions with target-derived neurotrophic factors can rapidly modulate the pattern of sympathetic activity and the effects of that activity on synaptic function. An understanding of these mechanisms will permit new approaches for developing therapeutics for cardiac pathologies.

How neural circuits form and are modulated by local and long distance signaling is a fundamental problem in neuroscience. Astrocytes are glial cells that provide one critical source of local signaling that regulates synapse formation and circuit function in the central nervous system (CNS). Less is known, however, about peripheral satellite glia that are found at the junctions between spinal cord neurons and peripheral sympathetic neurons. The neurons of the sympathetic ganglia provide critical neural control of peripheral functions, modulating the final output of central sympathetic drive, regulating peripheral organ function, and providing ongoing information to the CNS. Disruptions in the sympathetic circuit contribute to many human disorders, including neurological diseases such as Parkinson?s disease and dementia. Within the sympathetic ganglia, satellite glia enwrap neuronal cell bodies and the cholinergic synapses that form between spinal cord inputs and postganglionic neurons, yet how these glial cells contribute to the formation and function of the sympathetic circuit remains to be determined. We have shown that cultured satellite glial cells isolated from the superior cervical ganglion (SCG) promote the development of structural synapses and synaptic activity of sympathetic neurons, indicating a potential role in setting circuit activity. In addition, satellite glial cell activity has been implicated in the acute regulation of sympathetic-mediated cardiovascular processes, suggesting that these glia could provide short-term and long-term regulation in this system. Here we will perturb the activity state of ganglionic satellite glia in vitro and in vivo to test the hypothesis that these glial cells regulate the development and output of the peripheral sympathetic circuit by coordinating presynaptic and postganglionic neuronal properties. We will examine the effects of glia on the intrinsic, synaptic and functional properties of postganglionic sympathetic neurons in the absence of their spinal cord presynaptic partners by selectively enhancing glial activity with the hM3Dq DREADD (Designer Receptors Exclusively Activated by Designer Drugs) in vitro. We will then use DREADD-expressing mice to investigate glial effects at synaptic sites originating from spinal cord inputs in vivo. These experiments will allow us to define the cellular mechanisms of glial actions and to determine their effects on the output of the sympathetic circuit.