Eric Frank - US grants

For the brain to operate correctly, the connections among nerve cells must be appropriate. During embryological development, some of these "synaptic" connections may be made because the cells are genetically pre-programmed to make them. In other cases, however, the synaptic inputs to a nerve cell may influence which other cells it, in turn, contacts. A knowledge of how peripheral targets can influence synaptic connections in the brain is basic to an understanding of how nerve cells originally make and then refine and maintain their proper connections. Synaptic connections in the spinal cord between sensory and motor nerve cells innervating muscles, the simple knee-jerk reflex, provide an excellent experimental system for studying this problem at the level of single nerve cells. In the spinal cord, these connections initially form with a high degree of specificity. However, if a group of sensory cells is forced to innervate a different target during development, the synaptic connections formed within the spinal cord are appropriate for this new target. The experiments in this grant are designed to explore, using anatomical and microelectrode recording techniques, the following questions: When, during development, is a nerve cell specified to have a particular function? To what extent is this function determined by the previous history of the cell (its lineage) versus its present molecular environment? These studies should provide useful information concerning how synapses are made during normal development and even how they might be repaired after spinal injury.

As the nervous system develops, neurons become specified to make precise patterns of synaptic connections. Some aspects of neuronal phenotype are likely to be determined by a cell's lineage and its position within the developing embryo. Chemical signals present in a neuron's target tissue provide another mechanisms for influencing a cell's developmental fate. Neural activity can also shape the final pattern of synaptic connections. A knowledge of how these three mechanisms operate is basic to an understanding of how nerve cells originally make and then refine and maintain operate is basic to an understanding of how nerve cells originally make and then refine and maintain their proper connections. Synaptic connections in the spinal cord made by primary sensory afferent fibers innervating muscle and skin provide an excellent system for studying this problem at the level of single, functionally identified cells. Sensory neurons innervating muscle spindles form monosynaptic connections with spinal motoneurons, whereas cutaneous sensory neurons do not. The peripheral targets of sensory neurons are important determinants of the relative numbers of muscle vs. cutaneous cell types. A major aim of this proposal is to determine if this effect is caused by the selective survival of pre-determined sensory neurons, or if peripheral targets have an instructive effect on sensory neuron phenotype. Sensory fibers in the frog can regenerate after they are interrupted in the dorsal root. Although they re-innervate motoneurons with a high degree of specificity, they do not re- establish their long-fiber tracts within the dorsal columns. A second aim of this proposal is to see if neurite-promoting factors that work in vitro will also stimulate axon regeneration within the white matter of the spinal cord. Such studies may help in achieving regeneration in human spinal cord after accidental injury. Finally, we propose to continue our studies of the mechanism of presynaptic inhibition of sensory-motor synapses in the amphibian spinal cord. Synaptic transmission between muscle spindle afferents and motoneurons, the pathways that mediates the simple stretch reflex, is inhibited by stimulation of other sensory afferents. Previous work has shown that a major portion of this inhibition is presynaptic and is mediated by the neural transmitter GABA. However, activation of both GABAA and GABAB receptors inhibits synaptic transmission in this pathway. Experiments using pharmacological agents to block each of these receptor types should enable us to determine which type is responsible for the inhibition observed physiologically.

As the nervous system develops, synaptic connections between nerve cells are initially established in a highly specific manner. These initial contacts can then be modified to a limited extent, increasing their specificity still further. A knowledge of the functional and morphological correlates of synaptic development in the central nervous system is basic to an understanding of how nerve cells originally make and then refine and maintain their proper connections. Synaptic connections in the spinal cord between muscle sensory afferent fibers and motoneurons, connections which form the neuronal basis of the stretch reflex, provide an excellent experimental system for studying this problem at the level of single, functionally identified cells. In the frog's spinal cord, these connections form with a high degree of specificity and undergo only limited modifications thereafter. Vestibular inputs to these same motoneurons are also specific in adult frogs, but the manner in which this specificity develops in unknown. A major aim of this proposal is to compare the development of these two sensory inputs to gain insights into how specific synapses are formed. Sensory fibers in the frog can also regenerate after they are cut in the dorsal root, and they re-innervate motoneurons with a high degree of specificity. A continued study of the factors that influence the amount and specificity of this regeneration could help in achieving specific regeneration in human spinal cord after accidental injury. My objectives during the next five years are as follows: 1) Study the normal development of monosynaptic connections between muscle sensory axons and motoneurons at the level of individual functionally identified cells. 2) Extend these studies to the development of the vestibulospinal input to forelimb motoneurons. 3) Examine the effects of types of lesions on the extent of regeneration of sensory dorsal root axons into the spinal cord.

As the nervous system develops, synaptic connections between nerve cells are initially established in a highly specific manner. These initial contacts can then be modified to a limited extent, increasing their specificity still further. A knowledge of the functional and morphological correlates of synaptic development in the central nervous system is basic to an understanding of how nerve cells originally make and then refine and maintain their proper connections. Synaptic connections in the spinal cord between muscle sensory afferent fibers and motoneurons, connections which form the neuronal basis of the stretch reflex, provide an excellent experimental system for studying this problem at the level of single, functionally identified cells. In the frog's spinal cord, these connections form with a high degree of specificity and undergo only limited modifications thereafter. Vestibular inputs to these same motoneurons are also specific in adult frogs, but the manner in which this specificity develops in unknown. A major aim of this proposal is to compare the development of these two sensory inputs to gain insights into how specific synapses are formed. Sensory fibers in the frog can also regenerate after they are cut in the dorsal root, and they re-innervate motoneurons with a high degree of specificity. A continued study of the factors that influence the amount and specificity of this regeneration could help in achieving specific regeneration in human spinal cord after accidental injury. My objectives during the next five years are as follows: 1) Study the normal development of monosynaptic connections between muscle sensory axons and motoneurons at the level of individual functionally identified cells. 2) Extend these studies to the development of the vestibulospinal input to forelimb motoneurons. 3) Examine the effects of types of lesions on the extent of regeneration of sensory dorsal root axons into the spinal cord.

As the nervous system develops, neurons become specified to make precise patterns of synaptic connections. Some aspects of neuronal phenotype are likely to be determined by a cell's lineage and its position within the developing embryo. Chemical signals present in a neuron's target tissue provide another mechanisms for influencing a cell's developmental fate. Neural activity can also shape the final pattern of synaptic connections. A knowledge of how these three mechanisms operate is basic to an understanding of how nerve cells originally make and then refine and maintain operate is basic to an understanding of how nerve cells originally make and then refine and maintain their proper connections. Synaptic connections in the spinal cord made by primary sensory afferent fibers innervating muscle and skin provide an excellent system for studying this problem at the level of single, functionally identified cells. Sensory neurons innervating muscle spindles form monosynaptic connections with spinal motoneurons, whereas cutaneous sensory neurons do not. The peripheral targets of sensory neurons are important determinants of the relative numbers of muscle vs. cutaneous cell types. A major aim of this proposal is to determine if this effect is caused by the selective survival of pre-determined sensory neurons, or if peripheral targets have an instructive effect on sensory neuron phenotype. Sensory fibers in the frog can regenerate after they are interrupted in the dorsal root. Although they re-innervate motoneurons with a high degree of specificity, they do not re- establish their long-fiber tracts within the dorsal columns. A second aim of this proposal is to see if neurite-promoting factors that work in vitro will also stimulate axon regeneration within the white matter of the spinal cord. Such studies may help in achieving regeneration in human spinal cord after accidental injury. Finally, we propose to continue our studies of the mechanism of presynaptic inhibition of sensory-motor synapses in the amphibian spinal cord. Synaptic transmission between muscle spindle afferents and motoneurons, the pathways that mediates the simple stretch reflex, is inhibited by stimulation of other sensory afferents. Previous work has shown that a major portion of this inhibition is presynaptic and is mediated by the neural transmitter GABA. However, activation of both GABAA and GABAB receptors inhibits synaptic transmission in this pathway. Experiments using pharmacological agents to block each of these receptor types should enable us to determine which type is responsible for the inhibition observed physiologically.

DESCRIPTION: The correct operation of the nervous system depends on the formation of highly specific patterns of axonal projections and synaptic connections among nerve cells. To understand how this occurs, the approach taken in this proposal is to study the development of one well characterized neuronal circuit in detail, the simple monosynaptic stretch reflex. Sensory neurons supplying stretch-sensitive spindle organs in muscles (Ia afferents) project into the spinal cord where they make highly specific synaptic connections with particular subsets of motoneurons. The experiments proposed here examine how each aspect of the development of this sensory pathway is controlled: the initial differentiation of Ia afferents and the support of their survival by peripheral or central target factors, the molecular cues responsible for Ia axons to begin growing into the spinal cord, the guidance of these axons towards the ventral horn, and finally their arborization and the formation of functional synapses with motoneurons. These issues are addressed using two different experimental preparations of the developing chicken embryo: Organotypic cultures of spinal cord and sensory ganglia will permit the detailed study of the development of Ia projection patterns at a higher resolution than is possible in vivo. A parallel set of experiment on intact, surgically manipulated embryos will check if the results from culture also apply to more normal development in vivo. Finally an analogous organotypic culture system will be developed using embryonic mice to test the generality of these findings in a mammalian system. The six specific aims of this proposal are: (1) to determine if NT-3 instructs the differentiation of Ia sensory neurons, (2) analyze the relative roles of motoneurons and peripheral target muscle in controlling the number of Ia afferents, (3) analyze the cues within the spinal cord that guide Ia axons from the dorsal roots through the dorsal horn to the ventral cord, (4) determine the nature and identity of signals within the spinal cord that promote arborization of Ia axons in the ventral horn, (5) analyze the role of peripheral targets on the formation of specific patterns of synaptic connections between Ia axons and motoneurons, and (6) develop an analogous preparation of cultured spinal explants from embryonic mice.

Injuries to the brachial plexus result in the reduction or loss of use of the upper extremities due to damage to the brachial nerves and/or spinal roots. These injuries are usually the result of vehicle accidents or falls. Peripheral nerve grafts have been increasingly effective in restoring some motor and sensory function when only peripheral nerves are damaged or broken. Repair of damaged spinal roots is less successful, however, especially in restoring sensory function after damage or avulsion (breakage) of dorsal roots, which are the pathway of sensory axons from the periphery to the spinal cord. Sensory axons can regenerate within the root itself but do not cross into the spinal cord. When sensory input from the limb is drastically reduced or eliminated in this way, there are life-long disabilities for the injured person. The experiments proposed in this application are designed to develop potential therapies to restore sensory function after damage to dorsal roots. Several neurotrophic factors are already known to promote anatomical and functional regeneration of sensory fibers after dorsal root crush. Artemin, a member of the glial-derived neurotrophic factor family of neurotrophins, is especially effective in promoting the functional regeneration of both large, proprioceptive and small, nociceptive sensory axons after root crush. Artemin will be tested for its ability to restore specific and persistent sensory function following crush injuries of brachial dorsal roots in adult rats. Recovery of sensory function will be assessed using anatomical, behavioral and electrophysiological techniques. In a related project, a method for promoting regeneration after dorsal root avulsion will be developed. Many brachial root injuries result in complete breakage of the roots from the spinal cord (avulsions) rather than partial damage. In these cases, sensory axons must be guided back to the spinal cord before regeneration can occur. The cut ends of avulsed sensory roots will be surgically reattached to the spinal cord and treated with artemin to determine if sensory function can be restored. The goal of these experiments is to determine if artemin is sufficiently effective to warrant study for the repair of brachial root injuries in humans.

DESCRIPTION (provided by applicant): Most functional deficits after spinal cord injury are caused by the disruption of nerve fibers that project longitudinally and interconnect the brain and spinal cord. In principle, there are two possible strategies for re-building functional circuits to repair this loss of communication: nerve fibers that were not damaged can be stimulated to sprout collateral axons and build compensatory connections, and injured axons can be stimulated to grow across the lesion to reconnect with their original targets. Major progress has been made recently in promoting sprouting and regeneration of the corticospinal tract (CST), a major pathway for controlling movement. It is not known;however, if these sprouted and regenerated CST axons can re- establish functional synaptic connections. This proposal addresses that second step, determining if sprouting or regenerating CST axons can make functional synaptic contacts with their normal target neurons in the spinal cord. Recent studies have shown that genetic deletion of PTEN in CST neurons results in robust contralateral sprouting of their axons in the spinal cord following ablation of the contralateral CST and also promotes unprecedented regeneration of CST axons across spinal cord lesions. It remains unknown, however, if sprouted or regenerated axons can make functional synaptic connections. A major target of CST axons in mice are Clarke's column neurons, located in the C11 - L2 segments of the spinal cord, in close proximity to the CST. We propose to utilize in vivo electrophysiological approaches to assess the ability of re-growing CST axons form functional synapses with Clarke's column neurons. In the first aim, we will induce the axons in one CST to sprout into the contralateral spinal cord by interrupting the other CST via a unilateral pyramidotomy in PTEN-deleted mice. We will test if sprouted CST axons establish functional synaptic connections by selectively stimulating the CST while recording intracellularly from Clarke's column neurons. We can thus test if axonal sprouts can form functional synapses with appropriate synaptic targets in the spinal cord. In the second aim, the CST will be lesioned bilaterally by a complete spinal cord crush at T10. This surgical procedure interrupts all axons that project through the crush region. After allowing CST axons to regenerate through the lesion in PTEN-deleted mice, we will record intracellularly from Clarke's column neurons just caudal to the crush while stimulating the CST at cervical levels above the crush. These experiments will test if CST axons regenerating through the lesion are able to form functional synaptic connections below the lesion. Taken together, these experiments will allow us to assess an important functional aspect of sprouting and regenerating CST axons, namely their ability to form functional synaptic connections. These results should provide direct insights into designing therapeutic strategies for re-establishing corticospinal connections and promoting functional recovery after spinal cord injuries. PUBLIC HEALTH RELEVANCE: Spinal cord injuries cause a major loss of function because nerve fibers that connect the brain and spinal cord are disrupted at the site of injury. Major progress has been made recently in promoting re-growth of the corticospinal tract (CST), a major pathway for controlling movement, across the injured site, but it is unknown if CST nerve fibers can re-establish functional connections with appropriate nerve cells below the injury. The proposed experiments will use electrical recordings from mouse spinal cords to determine if re- growing CST nerve fibers can form functional connections with these nerve cells, thus providing insights into designing therapeutic strategies for promoting functional recovery after spinal cord injuries.