Avis H. Cohen - US grants

In this project the phenomena will be investigated which lead to functional recovery following spinal cord transection of lampreys. To lay foundation for this we will further characterize the locomotor central pattern generator (CPG) of the lamprey, since it is the CPG which must be successfully reconstructed to restore function. The physiology will include a comparison of oscillator stability between adult and larval cords, and an analysis of the role of the long tract coordinating fibers. Anatomical description of the tracts carrying coordinating fibers will be done to determine the pattern of distribution of the cells whose axons make up the tracts. Lampreys, larval and adult, will be spinal- transected. Their recovery will be further studied, and the behavior of the previously transected cords compared to controls. The regenerated neuronal processes will be mapped anatomically and compared to controls to determine whether the specific pattern of cell distribution correlates with functional recovery. Qualitative changes in segmental oscillators which result from spinal lesions will also be explored. A search for general principles of functional recovery which transcend the lamprey will be a major aim.

psychomotor function; neural conduction; neural information processing; neural initiation; bioperiodicity; invertebrate locomotion; neuroanatomy; spinal cord; membrane potentials; interneurons; psychophysiology; chordate locomotion; spinal cord surgery; Agnatha; spinal cord mapping; Hirudinea; mathematical model;

Movement by animals including man, requires precise timing to be smooth and coordinated. The neurons which control movement have been in existence for millions of years and if we can understand how they work we will have fundamental knowledge for understanding numerous aspects of movement from designing robots to rehabilitating stroke victims. The proposed study will focus on one of the oldest vertebrates known . the lamprey. Its nervous system has to provide accurate wave motions through pattern generating nerves which can be recorded in vitro. Also the lamprey undergoes a protracted larval phase during which the pace maker neurons must change cell firing rates to maintain the exact phase of the rhythm even though the neurons are growing longer and thereby taking longer to fire the muscle. The study will provide both basic biological data of the cell firing patterns during fictive swimming and also a mathematical analysis of the stable and unstable patterns and phase shifts. The work should tell us much about very basic events in the control of movement.

Previously, we showed fictive swimming in isolated lamprey spinal cords deviated from a uniform traveling wave. We asserted that the pattern of these deviations could be used to deduce functional properties of the intersegmental coordinating system. Using new statistical and theoretical methods, we are near a full description of the phase deviations and what they imply. In so doing, we have begun to generate statistical and theoretical tools to study changes in the spinal segments when interacting with descending and sensory inputs. By the end of year one we will begin to "put the system back together," that is, to reintroduce into the isolated spinal preparation other portions of the nervous system and sensorium. The goal is to begin to understand, in a vertebrate, how such systems are used adaptively by intact animals, and to deduce general principles of organization for such systems. Completion of the statistical and theoretical work involves: (a) simulations of the behavior of a chain of coupled oscillators with noise added. How does the noise propagate? How do the length and strength of the coordinating fibers affect the behavior? What statistical and time series methods are most appropriate for the analysis? We will then ask the following: 1. The phasic output of the reticulospinal (RS) neurons is said to be "in phase" with the motor output of the rostral segments, but this was with very few segments attached. If true, it could be disastrous since the relative phase angles of the activity of all 100 segments of the body must span 360 degrees of the cycle, and RS cells have powerful effects along the entire cord. We will use a preparation with brain stem and 50 segments to deduce: (a) What is the impact of the phasic activity from the RS nuclei upon the motor output of the spinal segments? Is the pattern more or less stable? Is it changed? (b) Using intracellular recording, what is the pattern of input from the spinal segments to the RS neurons? Is there some topographic map of the spinal segments along the nuclei? Or do all cells receive the same input? (c) Do the RS neurons have their own oscillations when activated with glutamate? What is the output from the reticular neurons to the spinal segments when a large complement of rostral and caudal segments provide input to the RS neurons? Is the RS neurons' output still phasic under these conditions? Does the output from the RS cells go to all spinal segments equally? Is it distributed across the entire cycle in all cells? (d) If phasic, how does the output from the RS nuclei interact with the coordination among the segments? This will be asked by modeling the interaction after the above data are collected. 2. If the brain and mechanosensory input both interact with the CPG, does the interaction change CPG output? (a) We will add mechanical forcing to the end of spinal segments with the brain attached to see how the output pattern is changed. This will also be done with the tail attached. Do the interactions stabilize or destabilize the vertebrate CPG? Do the brain/CPG/sensory interactions function as proposed in the cockroach to heighten responsiveness to unexpected perturbations and maintain some optimal frequency for the system? What are the functional consequences of the interactions? Finally, the overall goal is to see if we can establish principles of organization and function for motor systems that produce rhythmic movements.

This is a request for support for seven students and postdocs to attend the Bat Sheva Seminar on "Neurons and Neuronal Networks in the Motor System" at Hebrew University, Jerusalem, Israel, from March 14-15, 1993. The motor system controls the position of organisms and their movement in the external world by a complex assembly of neuronal networks. Intricate motor paradigms of movements and postural adjustments constitute almost all the behavioral patterns expressed in our daily life. Understanding of the motor system and its neural control mechanisms are therefore one of the most important aspects of modern neurobiology. This seminar is aimed at studying the motor system using an interdisciplinary research approach, starting from structural and functional aspects of single cells in the motor system, continuing with central neuronal networks, and ending with combined physiological and artificial intelligence studies. This approach will provide us better understanding of mechanisms involved in the execution of motor behavior. This seminar is an outstanding opportunity to facilitate advances in interdisciplinary training in basic neuroscience and motor control, and to bring together students and top-scientists from all over the world for a period of two weeks. Previous experience with Bat-Sheva seminars shows that it was very fruitful with good interactions among the participants and students, and this coming seminar seems to be more popular than the previous ones.***//

The lamprey has the property that its spinal cord spontaneously recovers from injury. There is ample evidence for fiber regrowth and functional recovery within a behaviorally relevant circuit, the central pattern generator(CPG) for locomotion. However, we present evidence to suggest the functional recovery is better described as dysfunction as both coordination and rhythmic stability are disrupted by the regenerated fibers. We propose to study the origins of this dysfunction. Specifically 1) we will obtain evidence after spinal injury as to the completeness of the behavioral recovery by recording muscle activity from swimming animals under unrestrained conditions. 2) The intrinsic coordinating system of the same animals will be characterized in vitro during fictive locomotion. Thus, we will compare the behavioral recovery with the CPG and regenerated coordinating system. The origin of any disruption in a given cord will be compared to the EMG data from the same animal to determine the contribution of sensory and descending input to the recovery. 3) The origin of the disruption in the "fictive"pattern will be correlated with an anatomical analysis of the neurons that have regenerated in that cord. Behavioral recovery will be assessed in the same animals continuously from one week until 10 months after injury. In some animals, the spinal cords will be isolated and tested at variable times beginning one week after injury, to include samples over the 10 months of recover. 4) The relative strengths of regenerated ascending versus descending fibers will be compared by examination of the response to mechanical forcing at rostral or caudal ends of the spinal cord. We hypothesize that a component of the disruptive changes originates from adaptive changes occurring over the course of an animal's life to match changing sensory input and motor output. To further test this hypothesis, we will examine the physiological and anatomical changes that occur in lampreys during their long transformation from ammocoete to adult and compare the results to those of regenerates. We will also assess the relative strengths of ascending versus descending coordinating fibers in ammocoetes and transformers using the same methods as those used for regenerates. Two modeling strategies will be developed in parallel to help us to understand the changes in both the coordinating system and the oscillators. These strategies both employ dynamical systems theory. A new type of model may allow more use of changes in the properties of the oscillators but lacks the property that the elements are themselves oscillators. This property will also be added to the new models and the two sytems that are generated will be compared.

Our research focuses on the production and control of locomotor behavior in the lamprey, with the goal of understanding behavior at the systems level. We propose three areas of investigation, two are continuations of previously begun projects, and one is new: I. Investigation of the structure and function of the spinal central pattern generator (CPG for locomotion, especially the intersegmental coordinating system. We will estimate functional coupling strengths in the CPG by fitting stochastic models to experimental data. Inhibitory baths of varying lengths will be used to study the relative strength of long versus short connections. Perturbations produced by bending the spinal cord with be used to help distinguish between ascending and descending coupling. Besides entraining the CPG, bending (forcing) elicts a slowly-decaying decrease in cycle period; we will search for a similar slow process in unforced data. We will extend the theoretical framework for entrainment by including long distance and non- relative-phase coupling; experimentally, we will determine how entrainment ranges vary with the number of active segments. Connectionist models of the CPG will be used to explore the dependence of intersegmental phase lags on the lengths and strengths of connections as well as the spatial variation in oscillator frequencies. We will analyze models of coupled heterogeneous populations of bursting neurons and explore the relevance of heterogeneity to intersegmental coordination. On a new experimental front, we will explore the use of optical dyes to record from groups of neurons in the CPG circuit, as well as to identify new classes of coordinating neurons. II. Normal locomotor behavior: integration of the components of the body to produce whole animal swimming. This is largely a continuation of work begun the last grant cycle. We will explore the role of the water in producing the traveling wave, the role of sensory inputs on the pattern, and the interaction between the brainstem and spinal cord. By combining all of these aspects of locomotor control, we are working toward a complete understanding of the production of an entire behavior in a vertebrate, and how all the parts of the organism and its environment combine and interact to produce that behavior. III. Longitudinal studies of the development and adaptability of the lamprey locomotor CPG. This is a new direction for the lab. The fictive swimming all sized of larval lampreys obtainable will be recorded, and the coupling strength among the segments estimated. If the motor pattern of the spinal cords of any size behaves very differently from the adults, new stochastic models will be used for the estimations. Also, we will raise animals under a variety of test to test the plasticity and adaptability of the CPG.

Our research focuses on the production and control of locomotor behavior in the lamprey, with the goal of understanding behavior at the systems level. We propose three areas of investigation, two are continuations of previously begun projects, and one is new: I. Investigation of the structure and function of the spinal central pattern generator (CPG for locomotion, especially the intersegmental coordinating system. We will estimate functional coupling strengths in the CPG by fitting stochastic models to experimental data. Inhibitory baths of varying lengths will be used to study the relative strength of long versus short connections. Perturbations produced by bending the spinal cord with be used to help distinguish between ascending and descending coupling. Besides entraining the CPG, bending (forcing) elicts a slowly-decaying decrease in cycle period; we will search for a similar slow process in unforced data. We will extend the theoretical framework for entrainment by including long distance and non- relative-phase coupling; experimentally, we will determine how entrainment ranges vary with the number of active segments. Connectionist models of the CPG will be used to explore the dependence of intersegmental phase lags on the lengths and strengths of connections as well as the spatial variation in oscillator frequencies. We will analyze models of coupled heterogeneous populations of bursting neurons and explore the relevance of heterogeneity to intersegmental coordination. On a new experimental front, we will explore the use of optical dyes to record from groups of neurons in the CPG circuit, as well as to identify new classes of coordinating neurons. II. Normal locomotor behavior: integration of the components of the body to produce whole animal swimming. This is largely a continuation of work begun the last grant cycle. We will explore the role of the water in producing the traveling wave, the role of sensory inputs on the pattern, and the interaction between the brainstem and spinal cord. By combining all of these aspects of locomotor control, we are working toward a complete understanding of the production of an entire behavior in a vertebrate, and how all the parts of the organism and its environment combine and interact to produce that behavior. III. Longitudinal studies of the development and adaptability of the lamprey locomotor CPG. This is a new direction for the lab. The fictive swimming all sized of larval lampreys obtainable will be recorded, and the coupling strength among the segments estimated. If the motor pattern of the spinal cords of any size behaves very differently from the adults, new stochastic models will be used for the estimations. Also, we will raise animals under a variety of test to test the plasticity and adaptability of the CPG.

DESCRIPTION: (Verbatim from the Applicant's Abstract) The larval lamprey exhibits axonal regeneration and functional recovery following spinal cord lesions. However, the PI has found that by altering the temperature at which animals are maintained, it is possible to produce animals that exhibit either functional or dysfunctional swimming behavior. Thus the lamprey offers the opportunity to study the nature and consequences of dysfunctional regeneration. She has further found anatomical differences between the regenerated systems under the two conditions, specifically in terms of the distribution of serotoninergic axons. Following transections, there is a dramatic increase in serotonin fibers in areas rostral to the lesion, and a dramatic loss in caudal segments. However under conditions that favor recovery, serotonin fibers are more abundant in the caudal segments. She hypothesizes that serotonin fibers are responsible for at least one form of the behavioral differences seen under the different temperature conditions. The PI now proposes to identify the sources of the increased serotonin innervation in rostral segments, and the fate of the lost serotonin in caudal segments. One possibility that will be explored is that the increases in caudal segments is due to collareral sprouting of descending serotonin systems. She will test this hypothesis by evaluating whether the serotonin fibers originate from the brainstem or from dorsal roots. The fate of dopamine fibers will also be evaluated. Other studies will evaluate the functional capabilities of the animals using a sophisticated stochastic phase model to evaluate physiological responses. This technique, which has been developed by the PI, provides a quantitative measure of the degree of coordination between segments. In the same animals, the regenerated fibers will be traced anatomically and using specific markers for GABA and glycine.

[unreadable] DESCRIPTION (provided by applicant): Locomotion is the product of neural output acting on muscles driving a mechanically complex body in an unpredictable environment. The lamprey is a simple, well-studied and relatively tractable vertebrate model with which to probe this neuromechanical system. We hypothesize that steady locomotion in a predictable environment requires only the central pattern generator (CPG) without the necessity of other input. We also hypothesize that in an unpredictable environment sensory feedback combined with strong intersegmental coupling is necessary. To investigate these hypotheses we will develop an integrated model, of lamprey swimming LAMPREYCOMP with Thelma Williams, from London, and researchers Philip Holmes and Alexander Smits from Princeton University. The model spans CPG, sensory feedback, muscle mechanics, body mechanics, and fluid mechanics, and is a full model of a complex behavior in a vertebrate, albeit a simple one. Cohen and colleagues will perform experimental studies of the CPG and its response to sensory feedback from spinal mechanoreceptors, skin and lateral line and will develop the component of LAMPREYCOMP that maps sensory input to motor nerve output. Work on muscle and body mechanics will be done by Holmes and Williams. Smits and Holmes will study the fluid mechanics of the swimming animals both experimentally and theoretically. The experimental work will include developing a mechanical analog, P-RAY, whose motion will be adjusted to reproduce that of live animals, allowing measurements of fluid motion and pressure variations along the body. The theoretical work will include developing the fluid dynamical component of LAMPREYCOMP, in consultation with C. Peskin, L. Fauci and colleagues. LAMPREYCOMP will be tested to insure that it reproduces the behavior of whole animal swimming, various reduced preparations and P-RAY. It will then be used to investigate the effect of manipulations, such as removing sensory feedback from spinal mechanoreceptors that are not experimentally possible. We also show that all components of the protocol are possible. Because the lamprey is a model system for all of vertebrate locomotion, our hypotheses and models will have broad implications for more advanced organisms in which such a complete approach is not presently feasible. [unreadable] [unreadable]

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