Brian Mulloney - US grants

The coordination of complex integrated movements is one of the most challenging tasks that faces the nervous system. An experimental approach that has proved very fruitful is the study of simple model systems. Crayfish have nervous systems where it is possible to identify a limited number of neurons that control swimming behavior. Dr. Mulloney has been a pioneer in the study of this nervous system. With NSF support he has found that the movement of each separate limb is driven by a specialized local circuit of neurons specific for that limb. Coordination of limb movements is controlled by a separate circuit of interneurons. This is a major advance in our understanding of movement control. With the renewal of this grant, Dr. Mulloney will be able to describe the synaptic organization of these local circuits and identify the properties necessary for normal coordination. He will also describe the cellular mechanisms by which inputs descending from higher centers can change the state of these local circuits to modulate swimming rate and direction. The results will have broad applications in the design of robot locomotion systems. The type of distributed control identified in this system is emerging as a general principle for the guidance of locomotion in multi-appendage systems.***//

The goal is to understand how neurons in pattern-generating circuits function and contribute to orderly movements. Local interneurons are the most abundant neurons in brains, but because they are small and difficult to record, little is known about their physiological characteristics. A small set of axonless, nonspiking local interneurons has been discovered in the crayfish that is part of the neural circuits that generate rhythmic swimming movements. These interneurons are identifiable, and large enough to permit experiments that explore their physiological properties and synaptic interactions. We propose three projects that will describe how these nonspiking local interneurons contribute to the swimmeret rhythm. To test the role of these interneurons in generating the swimmeret movements, individual interneurons will be perturbed or ablated while the nervous system is generating the swimmeret rhythm. The membranes of interneurons will be clamped to different potentials with a single-electrode voltage clamp to control their release of transmitter. Individual interneurons will be ablated by photoinactivation. Comparisons of the motor patterns generated before and during the voltage clamp or before and after ablation will reveal the contribution of these interneurons to the motor pattern in their own circuit and in the whole animal. To test the hypothesis that nonspiking local interneurons are premotor, and physiologically remote from sensory input, pairs of identified motorneurons, sensory neurons and interneurons will be injected with Lucifer yellow and HRP and then studied to describe and count their points of contact. Regions of apparent contact will be thin-sectioned for EM to locate any synapses. To analyze how these interneurons integrate synaptic currents, we will describe their passive electrical structure in a detailed compartmental model by combining measurements of their pulse-responses and step-responses with careful measurements of their anatomical structure. The predictions of this model will be tested with a single-electrode voltage clamp to measure synaptic currents and synaptic potentials from known sources. This analysis will test the hypothesis that "subunits" of synaptic integration exist in these dendritic structures.

The objectives of this research are twofold: to explain the relation of synaptic structure to synaptic strength, and to discover the developmental mechanisms that determine the relative strength of different synapses on a common postsynaptic neuron. Each of the sensory SR neurons in the crayfish abdomen, twenty neurons in all, synapse with an identified pair of target neurons in the last abdominal ganglion. The strengths of these synapses are graded in adult animals; posterior SR neurons make stronger synapses that anterior ones. The Specific Aims of this proposal are to describe quantitatively the structural basic of these physiological differences in synaptic strength, and to discover when and by what mechanisms these gradients of synaptic strength first appear during the animal's development. The postsynaptic response (PSP) of each of the pair of the target neurons to stimulation of each SR axon will be measured, and then a selected presynaptic SR axon and both target neurons will be filled with an intracellular marker, HRP. The synaptic contacts between the SR axon and each target neuron will be counted and mapped in the light microscope, and the structure of each contact examined in the electron microscope. By comparing the structures and locations of strong synapse with those of weak synapses, we will test three hypotheses that consider both presynaptic and postsynaptic factors: --that SR axons with strong synapses make more synaptic contacts with the target than do weak synapses. --that differential postsynaptic attenuation of PSPs arising at different sites on the target neurons causes the measured differences in synaptic strength. It is not known when these gradients of synaptic strength first arise. To discover whether this occurs during embryogenesis or during postembryonic growth, SR PSPs from the same target neurons will be measured in newly hatched crayfish. --If the gradients arise during postembryonic growth, the roles of synaptic competition among SR axons, of differences in their rates of addition of new synaptic contacts, and of growth of the targets themselves during normal development will be tested.

9514889 Mulloney The exploration of the neuronal bases for animal movements is one of the most challenging tasks that faces the neuroscientist. This task is made feasible through the study of simpler model systems, which are found among invertebrate animals. Dr. Mulloney has shown through his previous NSF support that in the crayfish small neural circuits in each body segment genepate the basic rhythmic movements, and that a separate neuronal coordinating system organizes the local movements into a coordinated pattern. With this additional funding, he will explore in more detail the specific means by which the reciprocally inhibitory interactions among identified neurons generate the basic oscillatory patterns of the local circuits. Mathematical simulation of the physiologically defined interactions by Dr. Mulloney will test the progress of the neurophysiological description and guide the conduct of further experimentation. The results from these studies will enhance our understanding of the neuronal bases for movements in all animals and have broad applications in the design of robotic locomotion systems.

Normal locomotion in vertebrates and anlaropods is a complex behavior, and the neural mechanisms that coordinate their limbs during locomotion are not known with any certainty. Rhythmic movements of the swimmerets of crayfish are known to be coordinated by neural networks in their segmental ganglia, and the analysis of the intersegmental circuit that coordinates movements of neighboring swimmerets holds promise of new insights into neural mechanisms controlling locomotion. This proposal presents new ideas about intersegmental coordination in the swimmeret system. These follow the recent demonstration that an older hypothesis, which postulated an increased excitability of local circuits in segments that normally lead each cycle of movements, was incorrect; these local circuits do not differ in their excitability. Instead, the intersegmental circuit that coordinates them is polarized. Preliminary physiological results show that axons of ascending and descending coordinating interneurons can be isolated separately, so the predictions of the coupled-oscillator model will be tested by selectively interrupting these axons and comparing the system' s subsequent performance with the prediction. The proposal also presents a new cellular model of the coordinating circuit that is based on published descriptions of individual coordinating units. The cellular model was developed by testing the performance of many alternative patterns of connections with components of the local circuits in each ganglion that coordinating interneurons might make. To test this new model, its responses to non-uniform excitation of local circuits will be compared with the swimmeret system' s responses to non-uniform excitation of individual ganglia. To test the cellular model' s predictions, the neurons that are targets of individual coordinating interneurons will be ma pped electrophysiologically. In experimental preparations, firing of each interneuron will be perturbed with injected currents. The system' s response to these perturbations will be measured and compared with simulations of the same perturbations in the model.

Locomotion in vertebrates and arthropods is a complex behavior, and the neural mechanisms that coordinate their limbs during locomotion are not well understood. The abdominal swimmeret system of the crayfish provides a relatively simple and accessible system to study the intersegmental coordination of movement. Within each segment of the tail, the swimmeret paddles show an alternating power stroke and a return stroke, as in most locomotor appendages, and the strokes of adjacent segments show specific timing relationships. There are small circuits of neurons (nerve cells) within each segment that have known identifiable single cells driving particular motor movements, and three types of interneurons that are involved in coordinating the movements. It is particularly useful that the crayfish ventral nerve cord and swimmeret system can be isolated in vitro and still show the intersegmentally coordinated swimming pattern. This project uses a combined physiological and computational modeling approach to define the dynamic circuitry for the pattern. A cellular model of the nerve cell physiological properties and network connections is used to predict the phase patterns of bursts of nerve impulse activity of the relevant neurons in the system. These predictions are compared with the activity patterns and connectivity in individual neurons recorded physiologically in the living circuit, including the small electrical potentials at synaptic connections between pairs of single neurons as well as the impulse traffic between segments. The relative importance of local and intersegmental pathways are assessed by stimulating individual coordinating interneurons when the intersegmental pathways are intact and when they are blocked. Intracellular dye fills anatomically confirm physiological connecting pathways. Refinements of the model by the experimental findings will yield a thorough description of how the dynamics of the interneurons contribute to stable intersegmental coordination, and how the properties of the synapses contribute to normal locomotion.
This work will have an impact beyond crustacean locomotion research, by leading to new insights about neural mechanisms that underlie the much more complex vertebrate circuits for locomotion. The integration of computational modeling with physiology will also have an impact on needed multi-disciplinary training of students and postdoctoral researchers in neuroscience and physiology in general.

DESCRIPTION (provided by applicant): The goal of this research is to understand in cellular terms how a central nervous system (CNS) coordinates the movements of different limbs during normal locomotion. Because of its modular segmental organization, the crayfish swimmeret system presents the opportunity to analyze in cellular terms the neural mechanisms that coordinate movements of different limbs. Three different coordinating interneurons arise in each of the four pairs of local circuits that control swimmerets, twelve pairs of neurons in all. Each of these neurons projects its axon to other segments in the CNS. When the CNS is actively expressing the swimmeret motor pattern, each of these interneurons fires a burst of impulses at a characteristic phase in each cycle. These spikes in these neurons are both necessary and sufficient for normal intersegmental coordination. These coordinating axons synapse with local commissural interneurons in other segments to form an intersegmental neural circuit. We will investigate how information about the state of the local circuit controlling a limb on one segment is encoded by these coordinating neurons and decoded again by the circuits that control other limbs. The Specific Aims of this proposal are: 1. To discover whether individual coordinating interneurons encode information independently, or whether correlated firing in different axons conveys important information. 2. To describe the transformation of coordinating information by local commissural neurons that are direct targets of coordinating axons. 3. To test assumptions of two cellular models of this coordinating circuit and to extend these models to incorporate new experimental results. These research projects will use simultaneous extracellular recording of spikes in coordinating axons and the motor output to sets of limbs, and microelectrode recording from coordinating neurons and commissural neurons in isolated crayfish CNS. They also require laser confocal microscopy, and computational analysis of circuit dynamics. This research will be a model for study of limb coordination during locomotion in other animals, including the spinal circuits that are critical to locomotion in mammals, including man.

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).

A major goal of neuroscience is to understand the dynamics of neural circuits. In most circuits, ignorance of intrinsic neural properties and synaptic organization limits our ability to understand the performance. This project exploits new discoveries of the synaptic organization of a circuit that coordinates limb movements during locomotion. An integrated program of experimental neurophysiology and mathematical theory will be used to explain how dynamics emerge from the interactions of intrinsic neuronal properties, synaptic dynamics, and synaptic organization. Using coupled-oscillator theory to guide neurophysiological experiments, and experimental results to constrain development of new models, a model of the circuit will be constructed and used to explore how intrinsic properties of different neurons and synaptic connectivity combine to produce the stable differences in phase of limb movements that are essential for effective locomotion. This modeling work will search for general principles governing dynamics in coordinating circuits. Because these circuits are central to many neural systems, the results should be applicable to many different systems. Many components of this project lend themselves well to undergraduate research. Undergraduate students will be heavily involved in summer research programs and Senior Honors Thesis projects. To augment their introduction to research, an advanced undergraduate course will focus on weekly research seminars by speakers who are successful members of underrepresented groups, followed by lunch and discussion with the speaker. To hone their presentation and communication skills, students will be encouraged to present their work in the departmental mathematical biology seminar series. Students will also be recruited from UC Davis REU programs that support students from economically disadvantaged backgrounds or from under-represented groups.

A major goal of neuroscience is to understand the dynamics of neural circuits. In most cases, ignorance of intrinsic neural properties and the synaptic organization of circuits limit our ability to understand a circuit's function or to cure disorders of the nervous system caused by its malfunction. Modulation by transmitters and hormones can change the properties of neurons and synapses in these circuits dramatically, but leave some features of the circuit's performance strangely unmodified. This project exploits new discoveries of the synaptic organization of a circuit in the crayfish central nervous system (CNS) that coordinates limb movements during locomotion. This coordinating circuit links four pairs of modular local circuits distributed in different parts of the CNS that control individual limbs and are active during locomotion. Periodic motor output from these local circuits is synchronized, but occurs with a stable difference in phase between neighboring circuits. The goal of this research is to understand and explain in cellular terms the encoding of information needed to coordinate these distributed circuits. Electrophysiological experiments and computational analyses will be used to study and model encoding of information by the circuit's key coordinating neurons.

How stable phase differences are maintained in the face of neuromodulation is a deep problem. For example, cholinergic modulation of this system changes the period and strength of its motor output, but does not affect the phase differences between neighboring circuits. Electrophysiological and pharmacological experiments will be used to analyze how cholinergic modulation tunes encoding so that phase remains constant when period changes. This research will generate insights and analytical methods that can be applied to similar systems in the brain stem and spinal cord, and to rhythmic neural circuits in the brain. Student participants will be involved in all aspects of the project. The results will also be directly applicable to the development of flexible and adaptive robotic devices.