Stanley B. Kater - US grants

The neuronal growth cone performs several key functions in the establishment, remodeling and repair of the nervous system. The growth cone is involved in elongation of the neurite, changing direction of its advance, recognizing cues from the molecular terrain, and in synaptogenesis. The present proposal will test the idea that critical aspects of the growth cone's performance rely upon partitioning duties to different sites; in particular, this work focuses on the fan-like filopodia that lead the advancing growth cone into new cellular environments. Recent high resolution microscopic observations have demonstrated that activities associated with even a single filopodium can substantially alter events within the growth cone proper. Such findings suggest a critical role for filopodia in several key developmental processes. This proposal tests a series of interrelated hypotheses which taken together suggest that filopodia serve multiple, quite different roles in neural development. We will investigate: The long standing proposal that a major role for filopodia is as antennae-like sensors of the environment. We will then investigate how the growth cone integrates filopodial inputs in order to distinguish between those environmental cues that produce minor course corrections in navigation (i.e., guidance) versus complete inhibition of growth cone motility (i.e., collapse). A rigorous pharmacological dissection will then defend the intracellular pathways underlying the amplification of filopodial calcium second messenger signals. These three investigations will provide insights into how information can be transduced from the environment into meaningful signals within the neuronal growth cone. Finally, we will investigate a process with the high resolution afforded by cell culture which has previously only been studied in situ and is based solely on histological evidence. That is, does an alternative mechanism of target localization exist whereby backbranches can be formed along the neurite? Such backbranches could then result in the generation of new neurites that reach the inappropriate target. This latter set of experiments investigates an important addition to the conventional view that the activities of the lead growth cone are the primary determinants of target localization. Taken together, the proposed research directly tests a series of related hypotheses that will elucidate the multiple roles served by filopodia at key times in neural development and regeneration. By employing newly developed preparations that allow high resolution analyses of several different functions and focussing these around fundamental issues of early development, these experiments will yield an integrated picture of the multiple contributions that filopodia make to the overall function of neuronal growth cones.

This proposal addresses those factors which influence neuronal form and connectivity. In particular the relationship between neurite outgrowth and the matrix of connections that a given neuron posseses will be investigated. We have found that adult neurons are responsive to transmitter in a unique way, that is transmitters can act as regulators of development and neuroplasticity by regulating the behavior of neuronal growth cones. In addition to other transmitter substances which alter growth cone behavior, serotonin, which was originally eluciated as regulating growth cone behavior in vitro, appears to play a role in neuroembryology. Furthermore, neuronal activity, that is the generation of action potentials, has a profound influence on neurite outgrowth. These results pose a class of questions regarding the autoregulation of neuronal form and connectivity. That is, does the activity of neurons bias their ability to grow and hence form synapses and conversely, once formed, do synaptic connections alter neuronal activity and in a feedback fashion, regulate the further synapse-forming potential of each component neuron? This activity could be either in the form of action potentials generated by an individual neuron or by release of neurotransmitter from adjacent neurons or from branches of the original neuron. In light of the results to date, we will evaluate the effect of electrical activity on synapse formation. We will also examine the effects of potential targets using cell culture methods addressing whether targets can alter neuronal activity and thereby alter growth and connectivity. We are now examining "classical" chemical synapses in culture to address factors governing chemical synaptogenesis. The majority of this work will be performed in cell culture where individual identified neuronal behavior can be observed without the complexity of the in vivo environment of the buccal ganglia. Complementary experiments in situ will parallel the work in culture, providing a whole animal context in which to relate the results observed in cell culture.

The proposed research examines roles for neurotransmitters and their cellular signal transduction mechanisms (electrical activity, intracellular calcium and other second messengers in the regulation of dendritic and axonal outgrowth in isolated individual and mitotic sister hippocampal pyramidal neurons. This model allows us to address several key problems not previously approachable including; 1) How are axons and dendrites different with respect to their outgrowth and its control by neurotransmitters, electrical activity and intracellular calcium? 2) Which of the neurotransmitters that provide input to pyramidal neurons in situ affect outgrowth in isolated neurons and where do they act on the neuron (growth cones, neurite shaft,soma)? 3) Based upon the fact that different classes of afferents provide different neurotransmitter influences in a sequential order during development, we will ask whether this is mirrored in the chronology of expression of outgrowth responses to the different neurotransmitters? 4) What are the cellular mechanisms by which neurotransmitters affect outgrowth? Are they the same mechanisms used in synaptic transmission? 5) What influence does cell lineage have on the expression of neuronal form and its modification by neurotransmitters? This question will be addressed using mitotically- related pyramidal neurons. The techniques used to address these questions include: culture of isolated pyramidal neurons: analyses of axonal and dendritic outgrowth rates, physical isolation of axons and dendrites: electrophysiological recording and stimulation: measurement of intracellular calcium levels with fura-2. The results obtained from these studies will provide important insights into how neurotransmitters and electrical activity might be involved in the generation of functional neuronal circuitry, its modification during adult life, and its degeneration in neurodegenerative disorders.

This Program Grant is intellectually centered around questions concerning the genesis of neuronal architecture. The experiments contained in the individual proposals are highly integrated with one another. Interactions between the growth cone and the environment that regulates neurite growth will be examined by several investigators. Several investigators will address the role of exogenous signals, while others will study the genetic specification of features and ion channels important to growth cone behavior. The ensemble of these various approaches will significantly advance our understanding of the mechanisms underlying the generation of the complex architecture of the nervous system. Bamburg will study the regulation of cytoskeletal elements during neuronal outgrowth, and examine how changes in the organization of these cyotskeletal elements are involved with the motile activity of the growth cone. Mykle's studies will define the role of calpains, which are important calcium dependent proteases that hydrolyze cytoskeletal proteins within the neuronal grow the cone. Kater's work will employ growth cones as assays for important features of the molecular terrain in developing systems. Here, the hypothesis has been made that surface-associated molecules convey information to the neuronal growth cone which is mediated by changes in intracellular calcium. Ishii's work deals with the interactions between nerve and muscle in the context of growth factors, which, as a general class, are known to be of great significance for the behavior of developing neurons. Beam, exploiting the powerful systems he has developed in muscle cells, will directly address questions of calcium channel localization, specification of calcium channel type, and calcium homeostasis with high precision. The material assets and intellectual cohesiveness provided by this Program Project will allow these investigators to use a broad spectrum of approaches to address a central problem of tremendous biological significance in an efficient, integrated and comprehensive fashion.

DESCRIPTION (from applicants' abstract) Given the rate at which evidence is amassing in support of astrocyte communication, it is important that the mechanisms underlying this communication be examined with the same rigor as was key to initially understanding the network behavior of neurons. Astrocytes are characterized in vivo as highly interconnected by gap junctions, and as having transmitter and receptor systems of unknown function. By reducing CNS complexity through established cell culture systems, both gap junctions and extracellular communication now have been found to serve roles in the propagation of calcium waves through networks of astrocytes. The passage of intracellular messengers through gap junctions has been recognized for some time as one mechanism for mediating calcium wave propagation. Recent results from this laboratory reveal the existence of an additional, and possibly dominant path: An extracellular message that is the focus of this proposal. We now know that astrocytes are capable of releasing an extracellular message which can be both necessary and sufficient for the sequential activation of neighboring astrocytes during the spreading calcium wave. The experiments in this proposal will identify the compound(s) serving as the extracellular messengers. They will examine the relative contribution of gap junction- versus extracellular message-mediated communication pathways for calcium wave propagation, and will test the hypothesis that these two paths represent parallel signaling routes. This work also will determine if the calcium transients that are the signature of the glial calcium wave have the function of evoking release of the extracellular message. Finally, the applicants will address a fundamental issue of the mechanisms of regenerative responses by individual astrocytes. They will ask whether an autocrine function, in which an astrocyte stimulates itself by the release of its own message, underlies the sequential activation of astrocytes along the course of the calcium wave. The questions addressed by this proposal are fundamental in themselves. The results of this proposal will therefore significantly enhance our ability both to interpret data already in the literature and affect the future experimental course of diverse research efforts.

DESCRIPTION (from applicant's abstract) Neuronal growth cones navigate by the precise execution of specific behaviors at a variety of decision points. Although a wealth of literature demonstrates that such growth cone events rely upon dynamic activities of both cytoskeleton and membrane constituents, our knowledge of underlying mechanisms is incomplete. Though cytoskeletal dynamics are being intensely studied, it has been difficult to directly investigate comparable activities of the plasma membrane. The last five years have seen major progress in our understanding of membrane dynamics in systems other than growth cones in large part due to the synthesis and implementation of the fluorescent dye FM1-43. This dye allows real time visualization of membrane events in living cells. The present proposal makes use of this powerful tool to test the overall hypothesis that endocytosis is a highly regulated process that is involved in multiple growth cone activities. Key publications have demonstrated that there are diverse, dynamic membrane stores within the growth cone and that the process of endocytosis is required for outgrowth. Pilot experiments with FM1-43 indicate that endocytosis is a surprisingly active process in neuronal growth cones. The first Specific Aim will test the hypothesis that endocytosis is activity dependent in the growth cone, and that electrical activity can regulate both endocytosis and the fate of endocytotic vesicles. The second Specific Aim will use defined guidance cues to evoke specific growth cone behaviors (such as turning, pausing, and stopping) in order to test the complementary hypothesis that endocytosis in growth cones is more specifically related to pathfinding activity. The third Specific Aim will test the hypothesis that the proximate mechanism controlling growth cone endocytosis and the fate of endocytotic vesicles involves changes in intracellular free calcium. The proposed studies will investigate endocytosis in growth cones with temporal and spatial resolution not previously possible and thus could well advance our understanding of membrane dynamics to the level already achieved for the cytoskeleton.