George Bittner - US grants

We will examine intercellular exchange in crayfish giant axons using techniques such as iontophoretic injection of electron dense tracers followed by ultrastructural observations of the location of the marked substances. From such studies, we expect to determine the cellular mechanisms by which proteins and other substances are mutually exchanged between crayfish giant axons or adjacent glia and whether such substances may be trophically important to the survival of severed giant axons. We expect that, compared to vertebrates, it will be easier to demonstrate the nature and functional significance of trophic exchanges of proteins or other substances in these CNS giant axons because of their large size and because of their highly developed exchange mechanisms, particularly when severed. Nevertheless, we expect that vertebrate axons use qualitatively similar cellular mechanisms for protein exchange, and that results from our model system will be directly applicable to vertebrates. A better knowledge of glial/neuronal or neuronal/neuronal trophic interactions is needed in order to develop better ways to treat or cure diseases such as multiple sclerosis or muscular dystrophies which may be caused by pathological changes in trophic relations in neuromuscular tissue.

Alcohol abuse (intoxication, tolerance development, physical dependency) is perhaps the most significant drug problem in America today. Alcohol has a primary effect via its actions on nerve synapses and it is probable that many of the behavioral changes associated with its abuse are due to its effects on mechanisms responsible for synaptic plasticity. Hence, the long term goal of this project is to define the mechanisms by which alcohol affects synaptic plasticity to produce intoxication, tolerance or withdrawal dependence. To attain this goal, crayfish neuromuscular junctions which exhibit much synaptic plasticity have been- developed as a model system to characterize the electrophysiological and biophysical mechanisms by which alcohol alters synaptit plasticity to produce behavioral changes. Our specific aims for this three yea: proposal are to characterize the pre- and postsynaptic mechanisms by which alcohol affects facilitation, post-tetanic potentiation, long-term potentiation, presynaptic inhibition, and postsynaptic sensitivity at neuromuscular junctions taken from control animals, from animals undergoing tolerance to chronic exposure, and from animals undergoing withdrawal dependence following chronic exposure.

This project is a collaboration between a chemical engineer and a physiologist, to develop new chemical systems to encourage nerve regeneration. This involves fundamental research in determining what substances cause nerve growth, and in building synthetic polymer substrates capable of supplying such substances when implanted into the human body. Similar basic research will focus on the problem of fusing cell membranes, so as to reconnect nerve systems. If this work is successful, it would have major medical benefits; furthermore, it might possibly be a step towards future work more relevant to the central nervous system, a topic which is difficult to work with but could involve very large long-term benefits to humanity.

Our long-term objective is to define electrophysiological and biophysical mechanisms which are responsible for the presynaptic plasticities of facilitation, augmentation, post-tetanic potentiation and long-term potentiation. All of these homosynaptic plasticides will be examined using the crayfish opener excitor neuron whose nerve terminals can be doubly penetrated a fraction of a space constant away from transmitter release sites whose evoked and spontaneous release can be recorded from large, identified postsynaptic muscle cells. Using this preparation, we propose to examine whether increases in calcium currents, decreases in several potassium conductances, and/or increases in internal calcium or sodium concentrations contribute to any or all of these homosynaptic plasticides. Electrophysiological and biophysical paradigms have been carefully designed to measure each of these ionic properties. Given the conservative evolution of many other cellular/molecular mechanisms (including axonal conduction and synaptic transmission), cellular/molecular mechanisms of these synaptic plasticities found at crayfish opener excitor synapses will almost certainly be found at mammalian synapses (including humans). Such knowledge is important because the synaptic plasticities of facilitation, augmentation, and post-tetanic potentiation are probably responsible for such behavioral phenomena as arousal and sensitization, whereas long-term potentiation may be the neuronal basis for learning and memory.

The slow rate, incomplete extent, and poor specificity of axonal regeneration is an important medical problem associated with significant dysfunction and loss of productivity. Our long term goal is to develop techniques using polymers to improve the rate, extent, and/or specificity of nerve axon regeneration in humans. To begin at attain this goal, in this application we propose to use a biopolymer hydrogel which we have recently developed as substitute for microsuture to hold together the stumps of severed nerved bundles (Specific Aim I). We expect that this hydrogel will produce greater regenerative success than microsuture in vivo because the hydrogel causes less tissue damage. We also propose to use a biopolymer (polyethylene glycol) in hypotonic salines with reduced calcium to fuse (reconnect) the severed halves of crushed nerve axons within minutes in vitro (Specific Aim II). Finally, we propose to use our recently developed biopolymer hydrogel to add mechanical strength to axons reconnected by polyethylene glycol in vivo (Specific Aim III). This biopolymer-based technique would increase the rate (and perhaps the extent) of reconnection of severed axons with denervated tissues. We will assess the ability of these biopolymer to improve the rate, extent, and specificity of regeneration of rat sciatic axons according to morphological criteria (axolemma and axoplasmic continuity in electron micrographs, diffusion of tracers). electrophysiological criteria (conductio of action potentials), and behavioral criteria (sciatic functional index). Given that traumatic injuries to nerve axons are rather common and axonal integrity is essential for proper neuronal function , successful development of one or more of these proposed techniques using biopolymer to improve axonal regeneration would have significant medical implications for humans.

DESCRIPTION (provided by applicant): A novel bioengineered technique to rapidly and permanently repair cut PNS nerves Our long term objective is to performance-optimize to translate for clinical use our novel and innovative technique to produce rapid and permanent repair of acutely and chronically severed mammalian PNS axons to restore the behavioral functions they mediated prior to severance. We apply a well-specified sequence of bioengineered solutions containing polyethylene glycol (PEG) and various anti-oxidizing or oxidizing agents to rapidly and permanently rejoin (PEG-fuse) completely cut-severed ends of rat sciatic axons as a model in vivo system so that PEG-fused axons are physiologically and morphologically intact through the lesion site and their stimulation restores behavioral functions mediated by intact sciatic nerves. Our physiological and morphological measures of axonal continuity are action potential conduction and intraaxonal dye diffusion across the lesion site and EM and immuno-histochemical analyses.. Our measures of behavioral restoration are Foot Fault Asymmetry test and Sciatic Functional Index. We retard Wallerian degeneration by cooling or cyclosporin A and then repair axons chronically severed for up 10 days by PEG-fusion. We performance optimize tissue (biocompatability) responses of acutely or chronically excised sciatic nerve segments that are used as inter- position autografts or allografts to PEG-fuse repair acutely or chronically cut rat sciatic nerves. Nerve severance is a common traumatic injury to PNS axons in humans. Various procedures currently slightly improve the number and specificity of PNS axons that reestablish connections following severance, but not outgrowth rate (~1mm/day) or time (weeks to years) for PNS axons to re-establish those connections. Target tissues may atrophy before re-innervation can occur. Consequently, target tissues are often non-specifically re-innervated and behavioral recovery is often poor. PEG-fusion dramatically improves the speed and efficacy of behavioral recovery following acute cut- or crush-severance of mammalian PNS axons. We can also retard axonal Wallerian degeneration of severed mammalian axons for up to 10 days to increase the time for successful PEG-fusion for up to 10 days post-severance so that nerve injuries do not have to be immediately treated. Our well-specified sequence of bio-engineered solutions and materials needed for PEG-fusion use only FDA-approved chemicals. Hence, our PEG-fusion technique developed on rat sciatic nerves as a model in vivo system should rapidly translate to clinical procedures. The results of our proposed R-01 have high potential for shifting the current emphasis of current research and clinical practice from devising procedures to enhance the results of slow axonal outgrowth to considering rapid repair by our novel PEG-fusion technique.