Albert A. Herrera - US grants

The proposed project is a neurophysiological and anatomical investigation of the mechanism by which unilateral denervation of a frog sartorius muscle causes increased synaptic effectiveness at neuromuscular junctions in the contralateral sartorius. This enhanced effectiveness is not due to increased synaptic size, which remains unchanged. Instead, transmitter release per unit nerve terminal length increases up to 8 times in contralateral muscles. Synaptic effectiveness will be assessed by estimating the safety margin for neuromuscular transmission and by using intracellular recording to measure quantal content and other release parameters. Light and thin section electron microscopy will be used to search for morphological correlates of synaptic effectiveness at single identified endplates whose transmitter release properties have been analyzed. Freeze fracture electron microscopy will also be used to correlate average synaptic effectiveness with intramembranous ultrastructure. The following hypotheses for the mechanism of enhanced release will be tested: 1) alteration in ultrastructure, especially in the number of arrangement of presynaptic active zones; 2) altered intraterminal Ca2+ buffering; 3) prolongation of the presynaptic action potential; 4) altered nerve terminal membrane surface charge; 5) altered Ca2+ channel properties. Contralateral denervation also causes an increase in polyneuronal innervation, i.e., the convergence of multiple presynaptic inputs onto the same postsynaptic site. The possibility will be tested that this increase is only an apparent one, resulting from the enhanced detectibility of previously present, but undetectably weak polyneuronal inputs. There is no evidence that contralateral denervation causes motor nerve sprouting as previously suggested in related experiments. This project should advance our understanding of how peripheral synapses are regulated and what role these regulatory processes play in normal neuromuscular function and disease-related changes. The major significance of this study, however, may lie in its contribution to understanding comparable synaptic plasticity in the central nervous system. Such plasticity seems to play an important role in the development and maintenance of the brain and its repair following injury.

The goal of the proposed project is to determine mechanisms regulating synaptic effectiveness and synaptic plasticity at neuromuscular junctions, using a combined physiological and morphological approach. Synaptic effectiveness will be assessed by estimating the safety margin for neuromuscular transmission and by using intracellular recording and voltage clamping to quantify neurotransmitter release and muscle fiber electrical properties. Light microscopy will be used to search for morphological correlates of synaptic effectiveness at single identified endplates whose release properties have been analyzed. Freeze fracture electron microscopy will be used to correlate average synaptic effectiveness with presynaptic intramembranous ultrastructure. The following naturally occurring and experimentally induced forms of plasticity will be examined: 1) differences in transmitter release between nerve terminals of the same size in different muscles; 2) the dependence of spontaneous and evoked release upon muscle stretch; 3) the increase in release seen in one muscle when its contralateral homologue is denervated; and 4) changes in release that depend on the size of a motoneuron's peripheral field. This project should advance our understanding of how peripheral synapses are regulated and what role these regulatory processes play in normal neuromuscular function and disease-related changes. The major significance of this study, however, may lie in its contribution to understanding comparable synaptic plasticity in the central nervous system. Such plasticity seems to play an important role in the development and maintenance of the brain and its repair following injury.

The frog neuromuscular junction will be used to study synaptic remodelling and the elimination of polyneuronal innervation during development. A new technique of vital staining with fluorescent dyes will be used to directly observe identified motor nerve terminals in living, anesthetized animals. Observations will be verified by conventional histology and electrophysiological recording. The first aim is to study motor nerve terminal remodelling in living adult frogs. There is substantial evidence that such remodelling occurs, but this evidence is from histological studies of fixed tissue. Through direct observation, this dynamic process can be described more thoroughly and the validity of commonly used histological criteria tested. The second aim is to observe nerve terminal remodelling as it is associated with the robust muscle growth that occurs in frogs during early adult life. Terminal remodelling takes two forms. First, terminals lengthen as target muscle fibers increase in diameter. Second, discrete new synaptic sites are added as fibers increase in length. The role of this remodelling in the maintenance of synaptic efficacy and in nerve-target trophic interactions will be explored. The third aim is to study developmental synapse elimination, using a unique adult preparation that offers several experimental advantages. Results will be used to test the hypothesis that competitive interactions between multiple nerve terminals at the same postsynaptic site are influenced by spatial proximity. The development and application of the innovative technique of in situ observation will significantly advance our understanding of synaptic plasticity at this accessible peripheral synapse. Based on historical precedent, results should be directly applicable to similar phenomena occurring in the brain and spinal cord. Such plasticity is likely to be of major importance in determining the ability of the nervous system to recover from injury or disease, and may be fundamentally involved in mechanisms of memory and learning.

The long-term objective of the proposed research is to understand the cellular and molecular mechanisms of neuronal plasticity in adult animals. Such plasticity undoubtedly plays a major role in the ability of the nervous system to recover from disease or injury, store information, and learn from experience. Specific experiments will focus on the regulation of nerve and muscle plasticity by androgen hormones. In several species, motoneurons and muscles related to sexual behavior have been shown to be regulated by levels of circulating androgens. The system to be studied consists of the flexor motoneurons and muscles of the forearm in male frogs (Xenopus laevis). These muscles are used to clasp females during mating behavior in such a way as to facilitate external fertilization. The system was chosen for its pronounced sexual dimorphism, wealth of background information, and easy accessibility for morphological, physiological, and behavioral studies. The first specific aim is to investigate the effects of androgens on muscle fibers. Hormone levels will be manipulated by surgical methods. Histology, histochemistry, and contraction measurements will be used to determined whether androgens have specific effects on muscle fiber number of contraction properties. The second specific aim is to test the effects of hormones on synaptic structure neuromuscular junctions. Histology and vital staining with fluorescent dyes will be used to ask whether androgens have effects on pre- or postsynaptic structure, and whether certain types of motor units are preferentially affected. The third specific aim is to test the effects of hormones on synaptic function at the neuromuscular junction. Electrophysiological and light microscopic techniques will be used to measure synaptic safety margin, neurotransmitter release levels, and postsynaptic electrical properties. It is expected that the results of this study will be of great usefulness in guiding future studies of neuronal plasticity in the brain.

In this continuing project, the frog neuromuscular junction will be used to study the relation between synaptic competition and synaptic remodelling during synapse elimination. The central hypothesis is that competition is the force driving remodelling in both developing and adult synapses. The experiments will employ new methods for in vivo observation of neuromuscular junctions as well as detailed electrophysiological studies of morphologically identified junctions. The first specific aim is to make repeated, in vivo observations of synapse elimination and remodelling in reinnervated junctions. Histology suggests that motor nerve terminals remain highly dynamic long after regeneration is complete. Physiology reveals no net change in polyneuronal innervation, however. The second aim is to correlate the tendency of terminals to persist or retract during synapse elimination with their physiological properties. Reinnervated junctions will be observed in vivo. When synapse elimination begins, muscles will be removed and electrophysiology used to determine the functional properties of identified nerve terminal branches. The third aim is to determine the morphological and physiological correlates of synaptic competition during a different form of synapse elimination. Using a preparation in which muscle fibers are innervated at two distant junctional sites, comparisons will be made between fibers where the two sites are innervated by the same or different motoneurons. These experiments may reveal whether plasticity at neuromuscular junctions conforms to Hebbian principles. Neuromuscular junctions are the best understood of all chemical synapses. Although they are certainly different from brain synapses, it is highly likely that cellular mechanisms of plasticity discovered at neuromuscular junctions will have wide applicability. A thorough understanding of synaptic plasticity is essential, for such processes probably underlie learning and memory and determine the ability of the nervous system to recover from injury or disease.

This proposal is for funding for a BioRad DVC-250 laser-scanning confocal microscope, together with associated camera, computer and printer equipment. This equipment will be used for imaging both living and fixed biological specimens, at high resolution, and in three dimensions. This rapidly maturing technology is now in very wide use, and is particularly suited to visualizing multiple antigens or stains, simultaneously, and their digital analysis. With the equipment system requested, we will be able to acquire microscopic images, process them with a computer, and print them out, at publication quality. While there are a number of other microscopes available on this campus (compound microscopes, Electron microscopes, etc.), there is no confocal microscope. The USC medical school houses a confocal system, but this is not available to labs on this campus. Two of the proposed major users now use confocal systems at other sites. Ko uses a system at the City of Hope Hospital (20 miles distant), which is not available to others. Warrior uses a system at U.C. Irvine (50 miles distant), which is available, but charges a $100/hour user fee. In short, the biological researchers here do not have regular, or open access to this important technology. We have examined three different confocal microscopes: the Nikon K2Bio, BioRad DVC-250, and the Meridian, and we have determined that the BioRad offers the best compromise of performance and cost, for our purposes. This institution (USC), has undertaken to provide 30% of the total cost of this equipment, as a cost share. This equipment system will be used by a group of Biological research labs at the University Park campus, of the University of Southern California (in Los Angeles). This group consists of five major users (Moses, Warrior, Tower, Herrera and Ko, the principal investigators listed), and five minor users (Bottjer, Manahan, McFall-Ngai, Thompson and Watts). All of the major users are currently supported by federal gr ants. While our research interests are somewhat disparate we all share a strong interest in using the confocal microscope to visualize structures and antigens in three dimensions in biological specimens. The Moses lab studies pattern formation and cell fate determination in the Drosophila compound eye. They will use the confocal system to colocalize gene products (such as Drosophila homologs of Transforming Growth Factor a, and its putative receptor), and to examine double and triple stained genetic mosaic tissues. The Warrior lab studies inter and intra-cellular movements in the developing Drosophila embryo. They will use the equipment to study germ cell migration, and the functions of the schnurri and DnudC genes in early development. The Tower lab studies development of the Drosophila chorion, and the regulation of DNA replication. They will use the system to quantitate DNA levels in the developing follicle cells. The Herrera lab studies developmental plasticity in frog neuromuscular junctions, and the action of steroid hormones on this structure. They will use the system to study the junction structures in living specimens, in which their function, and response to hormones, can be tested. The Ko lab studies neuro transmitter release and the function of the presynaptic terminal. They will use this equipment to explore the behavior and morphology of voltage coupled Calcium channels, using specifically binding toxins.