Lea Ziskind-Conhaim - US grants

The objectives are to study the inductive relationship between developing central and peripheral synapses, and the influence of these synapses on development changes in motoneuron properties. To ensure specific and long-term interaction between cells, differentiation of synapses is directed by gene expression as well as by influential signals passing in both directions across the synapse. Detailed information has accumulated describing the function of sensory-motor connection in adult vertebrates, but little is known about their development. Most knowledge comes from studies of developing central synapses in invertebrates, and non-mammalian vertebrates such as tadpole and chick. I will study the electrical and pharmacological properties of immature motoneurons in rat embryos, and investigate the roles of developing primary afferent-motoneruon contacts and peripheral nerve-muscle interactions on these properties. I shall study pattern and time course of development of spinal reflexes and the initial specificity of afferent-motoneuron contacts. Studies will be carried out in vitro, using isolated segments of thoracic spinal cord with or without their adjacent intercostal muscles. The small size of the embryonic spinal cord allows adequate penetration of oxygen and nutrients into the tissue. The advantages of using spinal cord-intercostal muscle explants are: 1) motoneurons are cultured in continuity with the muscles they normally innervate in vivo, and 2) we have previously described the chronology changes that occur during development of intercostal nerve-muscle contacts in vivo and in organ culture, and thus will be able to correlate these changes with the differentiation of motoneurons that innervate them. Maintaining these explants in the controlled environment of organ culture is valuable for isolating signals that influence motoneuron properties and the formation of central neuronal interactions. To broaden our knowledge about the way functional neuronal circuits develop, we must extend our information about the mechanisms involved in specificity of synapses during development. Such an understanding may give insight into mechanisms of long-term interactions between cells and reasons for their inability to re-establish these interaction following lesions in the central nervous system.

The objectives of this grant are to study the inductive relationship between developing central and peripheral synapses, and the influence of these synapses on developmental changes in motoneuron properties. To ensure specific and long-term interaction between cells, differentiation of synapses is directed by gene expression as well as by influential signals passing in antidromic and orthodromic directions across the synapse. Detailed information has accumulated describing the function of sensory-motor synapses in the spinal cord of adult mammals, but little is known about their development. Developing central synapses have been intensively studied in invertebrates and non- mammalian vertebrates such as tadpole and chick. This project will focus on studying the electrical, pharmacological and morphological properties of spinal motoneurons of rat embryos, and investigating the roles of developing sensory-motoneuron contacts and peripheral nerve-muscle interactions on these properties. The pattern and time course of development of spinal reflexes and the initial specificity of afferent motoneuron contacts in spinal cord of rat embryos will be examined. These studies will be carried out in vitro using isolated segments of spinal cord. The small size of the embryonic spinal cord allows adequate penetration of oxygen and nutrients into the tissue which remains viable for many hours. Thoracic segments of spinal cord with their adjacent intercostal muscles will be maintained in organ culture for up to 6 days. The advantages of using spinal cord-intercostal muscle explants are: 1) motoneurons are cultured in continuity with the muscles they normally innervate in vivo, and 2) we have previously described in chronological changes that occur during development of intercostal nerve-muscle contacts in vivo and in organ culture, and thus will be able to correlate these changes with the differentiation of motoneurons that innervate them. Maintaining these explants in the controlled environment of organ culture is valuable for determining the signals that influence motoneuron properties and the formation of central neuronal interactions. Understanding the mechanisms involved in specificity of synapses during development may give insight into mechanisms of long- term interactions between cells and reasons for their inability to re-establish these interactions following lesions in the central nervous system.

The objectives of this proposal are to characterize the changes in electrical and pharmacological properties of spinal motoneurons during embryonic development and after injury. We will determine: 1) the influence of newly formed central and peripheral synapses on motoneuron properties, and 2) the effects of sensory deprivation and axotomy on motoneuron differentiation. These studies will be carried out using two preparations: 1) isolated lumbar segments of rat spinal cord, in which functional sensorimotor pathways are preserved for at least 9 hours, and 2) spinal cord-muscle explants in which motoneurons are viable for 4-6 days. The isolated spinal cord of rat embryos is particularly suitable for studying neural and synaptic responses to known concentrations of ions and drugs. We have been using this preparation to study the pattern and time course of formation of excitatory and inhibitory synapses in spinal cord, and to examine the effects of the new synapses on motoneuron properties. The results of the proposed studies will establish the pattern of motoneuron differentiation in vivo, and will guide our future studies on motoneuron development in organ culture. Isolated segments of thoracic spinal cord with their associated intercostal muscles will be maintained in vitro for a period of several days. The advantage of using spinal cord-intercostal muscle explants are: 1) motoneurons are cultured in continuity with the muscles they normally innervate in vivo, and 2) we have previously described the chronology of changes that occur during development of intercostal neuromuscular junctions in vivo and in organ culture. We will thus be able to correlate these changes with differentiation of motoneurons that innervate these muscles. Maintaining these explants in a controlled environment provides valuable means for determining the extracellular factors that influence motoneuron properties and the formation of specific central neural pathways. Acquisition of motoneuron specific characteristics arises from interactions between intrinsic instructions and extracellular factors, but little is known about their relative contributions to differentiation of mammalian neurons. The proposed studies will extend our understanding about the way functional neural circuits develop, and the effects of newly formed pathways on neuron differentiation. This understanding should provide insight into mechanisms underlying long-term interactions between neurons in the central nervous system.

The objectives of this grant are to study the inductive relationship between developing central and peripheral synapses, and the influence of these synapses on developmental changes in motoneuron properties. To ensure specific and long-term interaction between cells, differentiation of synapses is directed by gene expression as well as by influential signals passing in antidromic and orthodromic directions across the synapse. Detailed information has accumulated describing the function of sensory-motor synapses in the spinal cord of adult mammals, but little is known about their development. Developing central synapses have been intensively studied in invertebrates and non- mammalian vertebrates such as tadpole and chick. This project will focus on studying the electrical, pharmacological and morphological properties of spinal motoneurons of rat embryos, and investigating the roles of developing sensory-motoneuron contacts and peripheral nerve-muscle interactions on these properties. The pattern and time course of development of spinal reflexes and the initial specificity of afferent motoneuron contacts in spinal cord of rat embryos will be examined. These studies will be carried out in vitro using isolated segments of spinal cord. The small size of the embryonic spinal cord allows adequate penetration of oxygen and nutrients into the tissue which remains viable for many hours. Thoracic segments of spinal cord with their adjacent intercostal muscles will be maintained in organ culture for up to 6 days. The advantages of using spinal cord-intercostal muscle explants are: 1) motoneurons are cultured in continuity with the muscles they normally innervate in vivo, and 2) we have previously described in chronological changes that occur during development of intercostal nerve-muscle contacts in vivo and in organ culture, and thus will be able to correlate these changes with the differentiation of motoneurons that innervate them. Maintaining these explants in the controlled environment of organ culture is valuable for determining the signals that influence motoneuron properties and the formation of central neuronal interactions. Understanding the mechanisms involved in specificity of synapses during development may give insight into mechanisms of long- term interactions between cells and reasons for their inability to re-establish these interactions following lesions in the central nervous system.

The objectives of this proposal are to characterize the changes in electrical and pharmacological properties of spinal motoneurons during embryonic development and after injury. We will determine: 1) the influence of newly formed central and peripheral synapses on motoneuron properties, and 2) the effects of sensory deprivation and axotomy on motoneuron differentiation. These studies will be carried out using two preparations: 1) isolated lumbar segments of rat spinal cord, in which functional sensorimotor pathways are preserved for at least 9 hours, and 2) spinal cord-muscle explants in which motoneurons are viable for 4-6 days. The isolated spinal cord of rat embryos is particularly suitable for studying neural and synaptic responses to known concentrations of ions and drugs. We have been using this preparation to study the pattern and time course of formation of excitatory and inhibitory synapses in spinal cord, and to examine the effects of the new synapses on motoneuron properties. The results of the proposed studies will establish the pattern of motoneuron differentiation in vivo, and will guide our future studies on motoneuron development in organ culture. Isolated segments of thoracic spinal cord with their associated intercostal muscles will be maintained in vitro for a period of several days. The advantage of using spinal cord-intercostal muscle explants are: 1) motoneurons are cultured in continuity with the muscles they normally innervate in vivo, and 2) we have previously described the chronology of changes that occur during development of intercostal neuromuscular junctions in vivo and in organ culture. We will thus be able to correlate these changes with differentiation of motoneurons that innervate these muscles. Maintaining these explants in a controlled environment provides valuable means for determining the extracellular factors that influence motoneuron properties and the formation of specific central neural pathways. Acquisition of motoneuron specific characteristics arises from interactions between intrinsic instructions and extracellular factors, but little is known about their relative contributions to differentiation of mammalian neurons. The proposed studies will extend our understanding about the way functional neural circuits develop, and the effects of newly formed pathways on neuron differentiation. This understanding should provide insight into mechanisms underlying long-term interactions between neurons in the central nervous system.

Summary Motoneuron differentiation and the formation of specific synaptic contacts arise from interactions between expression of genetic information, and epigenetic influences that are extrinsic to the neuron. The objectives of this proposal are to investigate and determine the mechanisms underlying neuronal differentiation and synaptogenesis in mammalian spinal cords. Studies are focused on the mechanisms by which newly formed central and peripheral synapses regulate the developmental changes in motoneuron electrical and pharmacological properties. Development-related increases in motoneuron excitability and in the efficacy of synaptic transmission will be studied using electrophysiological recording techniques. The importance of intracellular Ca2+ in increasing the efficacy of developing sensory-motor synapses will be determined by relating the developmental changes in spontaneous and pharmacologically induced changes in intracellular Ca2+ to physiological changes in synaptic transmission. Three preparations will be used: (l) isolated lumbar spinal cords with attached muscle nerves, (2) spinal cord slices in which sensory-motor pathways are preserved and neurons can be visualized for electrophysiological recordings and Ca2+ imaging, and (3) spinal cord explants in which motoneurons and synaptic pathways differentiate in the controlled environment of organ culture. Our recent studies established the pattern and time course of motoneuron development and synapse formation in utero, and will guide our studies on motoneuron development in organ culture. The proposed experiments are designed to increase our understanding of the roles of extracellular factors in synaptogenesis and neuronal differentiation. This knowledge will provide insight into mechanisms underlying long-term synaptic interactions between neurons in the central nervous system.

DESCRIPTION(Verbatim from the Applicant's Abstract): Spinal networks that produce locomotor-like rhythmic electrical activity are formed at early stages of neuronal differentiation in the rat spinal cord. Previous studies have suggested that each side of the spinal cord contains a group of spinal interneurons that form a network referred to as the central pattern generator, which produces rhythmic electrical activity independently of supraspinal and peripheral sensory inputs. The coordinated oscillatory potentials in opposite sides of the spinal cord are thought to be responsible for the left/right alternating hind limb movements. The primary goal of this application is to determine the developmental changes in the organization and functional integration of distinct neural networks that trigger rhythmic electrical activity and coordinate bilateral activity in the developing mammalian spinal cord. Real-time images of voltage-sensitive fluorescent dye will be used to record changes in electrical activity in various areas of the isolated spinal cord. The main objectives of our application are: (1) To characterize the changes in the spatiotemporal pattern of spontaneous coordinated rhythmic oscillations during embryonic and postnatal development, and study the properties of the potential underlying those activities. (2) To investigate the spatial organization and functional integration of rhythm-generating networks and networks that coordinate the oscillatory electrical activity between the ipsilateral and contralateral sides of the spinal cord. Mechanical lesions at specific sites and local pharmacological block of synaptic transmission will be used to test the role of specific pathways in the generation of locomotor-like activity. (3) To determine the functional relationship between neural pathways that mediate spontaneous and pharmacologically induced rhythmic oscillations at the onset of coordinated bilateral activity. Experiments will be carried out using complementary biophysical and electrophysiological approaches in thick spinal cord slices in which neural networks are preserved. These studies will increase our understanding of the mechanisms underlying the establishment of rhythm-generating networks and the complex functional integration of rhythmic activity that results in bilateral coordinated activity. The findings will be valuable for gaining insight into factors that might regulate long-term cellular interactions and synaptic plasticity in mature neural networks.

[unreadable] DESCRIPTION (provided by applicant): The autonomous spinal networks that generate movements in vertebrates are referred to as central pattern generators (CPGs). The principal neurons in the locomotor circuitry include ipsilateral rhythmogenic interneurons, (rhythm generators), commissural interneurons that coordinate the rhythms between the left-right sides of the spinal cord (rhythm coordinators) and motoneurons. The integrated activity of these neuronal populations produces rhythmic excitation of motoneurons during hindlimb movements in walking vertebrates. The primary challenge in studying the cellular and synaptic mechanisms underlying rhythm generation in the locomotor circuitry is the identification of interneuronal populations that comprised integral components of rhythm-generating and rhythm-coordinating networks. To overcome some of the technical difficulties identifying locomotor-related interneurons in the isolated spinal cord, transgenic mice have been used with the intention of characterizing interneuronal populations based on their unique gene expression. Recent studies have documented that ventral neurons can be divided into five domains of specific genes that represent physiologically distinct neuronal populations with defined functions in motor behavior. .Such genes have been widely used to express the reporter gene green fluorescent protein (GFP), giving rise to visually identified neurons that can be targeted for repeated electrophysiological, morphological and immunohistochemical studies. In this proposal two lines of GFP positive transgenic mice will be used to study the mechanisms of rhythm generation in ipsilateral excitatory interneurons expressing the HB9 protein, and inhibitory commissural interneurons that synthesize the enzyme GAD67. The objectives of this proposal are: (1) to test the hypothesis that the Hb9 and GAD67 interneurons are integrated in rhythm-generating and rhythm- coordinating networks, respectively and (2) to test the hypothesis that different synaptic and cellular mechanisms underlie rhythm generation in these interneurons that perform different functions in the locomotor circuitry. Understanding the mechanisms that modulate the patterns of locomotion rhythms in functionally identified interneurons in the isolated spinal cord that is disconnected from descending voluntary control, will provide important insight into possible therapeutic strategies of activating undamaged neurons in rhythm generating networks of patients with spinal cord injuries. [unreadable] [unreadable] [unreadable]