William B. Kristan - US grants

Many of the features in the ontogeny of behavior are common to all animals with a central nervous system. For instance, spontaneous coordinated movements appear before reflexes can be elicited in many animals, from insects and annelids through frogs, chicks and humans. The spontaneity has been attributed to the establishment of interneuronal connections to motor neurons before the sensory to interneuronal connections form. By using a relatively simple animal, the leech, in which both the structure and function of individual neurons has been well characterized, and which has a nervous system that is accessible for cellular studies throughout development, we can determine the cellular basis for such behavioral observations. We propose to study the neuronal basis for the development of two leech behaviors, reflex shortening and swimming. Using intracellular microelectrodes, we will record from identified sensory neurons, interneurons and motor neurons when these behavioral acts appear and are elaborated. We will characterize their individual electrical properties, particularly their ability to generate action potentials. We will also determine when they first make synaptic contacts, and follow the progress of the synaptic properties until they acquire all their adult-like features. In addition, we will fill the neurons with dyes, to characterize at both the light and electron microscopic levels the outgrowth of the neuronal processes and the ways in which processes of different cells come together to make synapses. Once we know the normal order of developmental events, we will perturb that order by cutting nerves, ablating target tissues, and by killing individual neurons to establish the rules by which neurons connect to one another to form circuits responsible for producing behavior. Hopefully, these studies will provide insight into the behavioral development of other animals, and will also provide a basis for studying the molecular mechanisms for the formation and maintenance of synapses in the development of behavior.

The ultimate goal of this project is to find the physiological, morphological and biochemical changes responsible for the recovery of neural and behavioral function after a neurological lesion. The results to date suggest that recovery of swimming behavior in the leech is accomplished by strengthening pre-existing synaptic connections secondary to sprouting of the swim-related neurons, possibly of those utilizing or responding to serotonin as their transmitter. In order to interpret these results unambiguously, we must first have a complete neuronal understanding of the swimming behavior. Hence, experiments will be performed both to characterize normal function and to show how function is restored after a lesion. Specifically, we will ask five questions: 1. Do newly discovered interneurons provide a complete explanation of the swimming motor pattern generation? 2. By what neuronal mechanisms does serotonin affect swimming pattern generation? 3. How does swimming relate to other leech behaviors? 4. How does the lesion trigger sprouting? 5. Can sprouting account for the neuronal changes responsible for the behavioral changes? To answer these questions, we must use physiological, anatomical, and pharmacological techniques. To complete the characterization of the swim pattern generator, we will record intracellularly from identified neurons to test the strength of their interconnections and, if necessary, search among the unidentified cells for neurons with pattern generating properties. We will apply serotonin and serotonin inhibitors to the ganglion while recording from pairs of swim-related neurons, to establish the cellular mechanism for the modulatory function of serotonin. We will use a variety of physiological anatomical techniques to find the trigger for neuronal sprouting and to test the importance of sprouting in functional recovery.

We propose to examine behavioral interactions at the level of single, identified neurons in the nervous system of the medicinal leech. We will first show how various behaviors interact by determining the probability of eliciting each of four different behaviors--swimming, walking, shortening and local bending--in response to mechanosensory stimuli. The variability in the responses will provide us with a measure of behavioral set of the animal. We will then show how the four behaviors interact by delivering pairs of stimuli, either simultaneously or in sequence, which individually would elicit one of the behaviors very reliably. If the pairs of behaviors are mutually exclusive, these studies will measure the animal's behavioral choice; from different pairings, we will determine the animal's behavioral hierarchy. The behaviors may prove to be compatible, so that elements are noninterfering or coordinated. It appears that swimming and crawling are coordinated, that local bending does not interfere with crawling or swimming, and that all other behaviors are organized into a hierarchy. We will also determine to what extent these behavioral interactions are modified by neuromodulators, particularly serotonin, and by learning. In parallel studies, we will characterize the cellular and network properties responsible for initiating and generating each of the four behaviors. This process is nearly complete for two of the behaviors, swimming and local bending, and is well begun for the other two. We will impale and record from two or three neurons simultaneously in semi-intact, behaving leeches or in isolated nerve cords. We will test the neuronal characterizations for the completeness by comparing the activity pattern recorded in the animal to a digitized model network, under both normal and perturbed conditions. The perturbations will be: 1) polarizing one to three neurons at a time, or 2) eliminating the interactions between neurons altogether by photoablation. We will then determine the interactions between neurons in the four behavioral circuits, to find the neuronal basis for the compatibility, coordination, or mutual exclusivity of the behaviors. Based on this information, we will be able to determine the site and nature of neuronal changes responsible for the modification of these interactions by neuromodulators and by learning. Such neuronal mechanisms are likely to be found in all animals, including humans, when behaviors interact.

We propose to examine behavioral interactions at the level of single, identified neurons in the nervous system of the medicinal leech. We will first show how various behaviors interact by determining the probability of eliciting each of four different behaviors--swimming, walking, shortening and local bending--in response to mechanosensory stimuli. The variability in the responses will provide us with a measure of behavioral set of the animal. We will then show how the four behaviors interact by delivering pairs of stimuli, either simultaneously or in sequence, which individually would elicit one of the behaviors very reliably. If the pairs of behaviors are mutually exclusive, these studies will measure the animal's behavioral choice; from different pairings, we will determine the animal's behavioral hierarchy. The behaviors may prove to be compatible, so that elements are noninterfering or coordinated. It appears that swimming and crawling are coordinated, that local bending does not interfere with crawling or swimming, and that all other behaviors are organized into a hierarchy. We will also determine to what extent these behavioral interactions are modified by neuromodulators, particularly serotonin, and by learning. In parallel studies, we will characterize the cellular and network properties responsible for initiating and generating each of the four behaviors. This process is nearly complete for two of the behaviors, swimming and local bending, and is well begun for the other two. We will impale and record from two or three neurons simultaneously in semi-intact, behaving leeches or in isolated nerve cords. We will test the neuronal characterizations for the completeness by comparing the activity pattern recorded in the animal to a digitized model network, under both normal and perturbed conditions. The perturbations will be: 1) polarizing one to three neurons at a time, or 2) eliminating the interactions between neurons altogether by photoablation. We will then determine the interactions between neurons in the four behavioral circuits, to find the neuronal basis for the compatibility, coordination, or mutual exclusivity of the behaviors. Based on this information, we will be able to determine the site and nature of neuronal changes responsible for the modification of these interactions by neuromodulators and by learning. Such neuronal mechanisms are likely to be found in all animals, including humans, when behaviors interact.

My colleagues and I would like to study the neuronal basis of complex behaviors in the nervous system of the medicinal leech. We will first complete the characterization of the interneuronal circuits responsible for four leech behaviors: local bending, shortening, swimming and crawling. We will then show how these circuits overlap and interact in determining the animal's response to mechanosensory stimulation. We have found recently that one of these behaviors, local bending, is produced by interneurons arranged in a distributed circuit. By using the back-propagation of error algorithm in neural network models, we have produced simulations which have many of the characteristics of the real interneuronal circuits. We propose to continue this approach, to determine whether the other three behavioral circuits are also distributed; there are strong indications that at least one of them is. In particular, we will study the mechanisms by which the nervous systems chooses among several possible behaviors, how the same interneurons contribute to several different behaviors, and how these networks are modified by learning and by embryological development. We will perform these experiments by recording intracellularly from neurons while the behaviors are being performed, then characterize the connections among the interneurons responsible for the behaviors. We will then use the neural network simulations to ask whether the identified circuit is complete and to suggest the properties of any undiscovered neurons. We will also use these neural networks to test whether there are other, possibly better, networks that can perform the same behaviors. Distributed networks are difficult to conceptualize, because every neuron contributes a little to every behavior and because some of the connections made by every neuron are inappropriate for each behavior. The neural network modeling techniques have given us a way to think about distributed networks and to make testable predictions about them. Such networks have been proposed for perception and motor control in more complex animals, but our work is the strongest indication that distributed networks control behaviors in a simple invertebrate. We are confident that our work can help to explain how such distributed systems process information and coordinate complex behaviors. Also, because we can measure many of the relevant neuronal parameters at the level of identified neurons, we should be able to help to refine the models and make them more biologically realistic. I believe that there will be major insights in the next decade into the ways that brains function as complex systems, and I feel that our integrated physiological and computational approach can contribute significantly to this understanding.

Partial support is requested to defray room and board costs for graduate students attending the 1993 West Coast Regional Developmental Biology Conference. The conference will be held on March 25-28 at the UCLA Conference Center at Lake Arrowhead, California. The program includes 24 speakers, who were chosen both for their scientific excellence and to provide a full mix of gender, race, and degree of professional advancement, as well as to provide information of interest in both animal and plant development. The topics to be addressed in the five formal sessions are: ?1! Asymmetric Cell Divisions in Differentiation, ?2! Control of Pattern Formation, ?3! Steroid Hormone Action on the Developing Nervous System, ?4! Influences of Bacteria on the Development of Higher Plants and Animals, and ?5! Determinants of Cell Fate. The conference will also include a keynote address to be given by Dr. Mark Konishi on the development of bird song, and all attendees will be encouraged to present their own work at a formal poster session.

This proposal seeks to explain movements of the body of the leech, Hirudo medicinalis, in terms of the forces generated by its muscles and by the arrangement of these muscles in the body wall. In addition to moving the animal, these muscles also produce a stiff tube which constitutes a "hydrostatic skeleton". There is no general theory for such a skeleton that would allow us to predict the shape of the animal's body from the activity pattern of its motor neurons. Therefore, the goal of this proposal is to use a combination of experimental measurements and numerical simulations to derive a quantitative description of hydrostatic skeletons in general and of the leech in particular. This study will proceed in three steps: 1. Describing leech movements in biomechanical terms. Segmental volumes, muscular tensions, geometric relationships of the muscles, internal pressures, and fluid flows will be determined. 2. Developing an analytical model of hydrodynamic skeletons. The current best model will be expanded to produce more realistic body forms, using the physical measurements obtained above. 3. Refining the model by comparing its performance to behaviors. Measurements during the production of simpler behaviors (bending and shortening) will be used to determine parameters which will tested by the model's ability to produce the more global behaviors (swimming and shortening). Such a model should be applicable to other hydroskeletons as well.***

DESCRIPTION (provided by applicant): The objective of this research is to understand how developing neurons form synapses onto particular neurons to form a functional neuronal circuit. These mechanisms will be studied in a relatively simple nervous system--that of the medicinal leech--because many of its neurons can be identified even before they start to grow the processes that will be the sites of synapses. In addition, a great deal is known about the morphological and physiological properties of these identifiable neurons, including the role that many of them play in generating behavior. Experiments will focus on the development of connections in two well-defined neural circuits: the connection between a pair of identified motor neurons, both of which innervate the same muscles in the body wall, and the connections in a reflex pathway that includes a sensory receptor, an effector neuron, and an intervening layer of a small number of interneurons. The generation of specificity will be approached using the following techniques: detailed morphological examinations of the spatial relationship between neuronal branches as the contacts are first established; physiological recordings from the synaptic connections as they form; and ablations of single neurons, of branches of neurons, or of peripheral targets, to perturb the environment in which the neurons are developing. These experiments will determine which mechanisms are operating in this well-defined system. For example, we will test whether neurons form synapses with any other neurons to which they are sufficiently close at a particular time; whether potential synaptic targets respond to environmental signals in a way that puts their branches in close proximity, even though the two neurons are behaving independently of one another; whether competition occurs for synaptic space between neurons that might be eligible to form synapses onto the same neuron; how the peripheral target of a neuron can control which synapses it will accept; and whether electrical activity is important for establishing or maintaining synaptic contacts. Experiments on other species, including higher mammals, have suggested that these same mechanisms control synaptic specificity in humans. The experimental tractability of the embryonic leech nervous system makes it possible to examine how the mechanisms interact to form a functional neuronal circuit among a number of identified neurons in a single species, thereby shedding light on how functional neuronal circuits are established in our own nervous system.

The International Congress of Neuroethology has met at three-year intervals since 1986, to bring together researchers working on the neural basis of behavior in vertebrate and invertebrate animals. The huge range of behaviors shown by many sorts of animals is a challenge to explain, and this diversity also can be used to illuminate basic principles of the organization and design of brains in general. It is productive, for example, to study a specialist in a particular kind of behavior such as bat echolocation, or a relatively simple system such as leech swimming, because detailed comparisons of the networks of nerve cells in brains often reveal unexpected mechanisms that need to be incorporated in our thinking about the complexity of brain function. The conference format is designed to emphasize talks on behavioral specializations, and on comparing common neural mechanisms across animals, with other talks on technical advances such as optical imaging and computational techniques for studying brain function. This award will provide partial support for student travel to the meeting.
This meeting will have an impact across neuroscience, from cellular approaches to behavior, and will also foster the careers of young investigators, and so have an impact on the infrastructure of neuroscience.

DESCRIPTION In many invertebrate and vertebrate species serotonin plays a relatively conserved neuromodulatory role, affecting motor output and the probability of performing different behaviors. In humans, serotonin has been strongly associated with depression. Yet, there is no clear idea of how it achieves its behavioral effects nor what is the natural input to the serotonergic system. A comprehensive description of the natural pathway through which the serotonergic system is activated could shed light on its physiological action. In the leech the serotonergic system is composed of a few types of neurons readily accessible to electrophysiological study. The major source of serotonin in this system, the Retzius (Rz) neurons, receive strong excitatory input from the pressure mechanosensory (P) neurons. The P-Rz connection is a polysynaptic pathway which spans the entire nerve cord and allows to channel mechanosensory input from any site of the body into a wide serotonergic output. The collaborators will investigate the organization of the neuronal pathway that mediates the P to Rz interaction. Their goal is to give a full description of the neurons that participate in this pathway. This will allow them to analyze how the sensory input channel produces fast behavioral responses, and at the same time activates modulatory pathways that change the behavioral responsiveness of the organism.

9987614
Terry Sejnowski - University of California, San Diego
IGERT: Graduate Training Program in Computational Neurobiology

This Integrative Graduate Education and Research Training (IGERT) award supports the establishment of a multidisciplinary graduate training program of education and research in computational neurobiology. The goal is to train a new generation of scientists and engineers with a broad range of scientific and technical skills who are equally at home measuring large-scale brain activity, analyzing the data with advanced computational techniques, and developing new models for brain development and function. This integrative training program is centered in the Department of Biology at UCSD and the Salk Institute, but includes faculty members from physics, chemistry, psychology, cognitive science, electrical engineering, computer science, and mathematics, as well as from biology and neuroscience. The training program will give all students hands-on experience in a wide range of advanced experimental and computational techniques through collaborative research between laboratories, industrial internships, and the opportunity to pursue research abroad. The faculty will participate in outreach programs to encourage and prepare underrepresented minorities for a career in computational neurobiology. Research areas in the training program include: (1) synaptic growth and plasticity; (2) neural dynamics; (3) neural population coding; (4) visual perception and memory; (5) stochastic learning algorithms; and (6) functional brain imaging.

IGERT is an NSF-wide program intended to meet the challenges of educating Ph.D. scientists and engineers with the multidisciplinary backgrounds and the technical, professional, and personal skills needed for the career demands of the future. The program is intended to catalyze a cultural change in graduate education by establishing new, innovative models for graduate education and training in a fertile environment for collaborative research that transcends traditional disciplinary boundaries. In the third year of the program, awards are being made to nineteen institutions for programs that collectively span all areas of science and engineering supported by NSF. The intellectual foci of this specific award reside in the Directorates for Biological Sciences; Computer and Information Science and Engineering; Social, Behavioral, and Economic Sciences; Mathematical and Physical Sciences; Engineering; and Education and Human Resources.

DESCRIPTION (provided by applicant) This research will be performed primarily in the laboratory of Dr. Szczupak in the University of Buenos Aires, Argentina as an extension of the NIH grant RO 1 MH 43396. The long-term objective of the project is to understand the structural configuration of sensory-motor networks. Specifically the aim of the present proposal is to reveal the role of a specific type of neurons which operates entirely in a graded mode. This type of neurons, recognized as non-spiking neurons, are different than the majority of cells in the nervous system in that they can operate as a highly compartamentalized structure subserving different networks due to the fact that propagation of signals is not supported by regenerative processes like spike firing. Investigation of their functional role in the simple nervous system of the leech will enable to assess the physiological rote of these type of neurons and illustrate about their function in other systems.

[unreadable] DESCRIPTION (provided by applicant): The purpose of the Systems and Integrative Neurobiology (SAIN) Training Grant is to provide a coherent and diverse training program for students interested in the neuronal basis of perception and behavior. This training program is in its 20l year, and currently supports 11 students, although 12 slots were approved for funding. We are requesting an increase to 15 funded slots, based upon the increase in number of participating faculty and the increase in number of highly talented applicants in this area of biological research. Although administered through the Division of Biological Sciences, this program is run by faculty from additional departments on campus (Psychology, Physics, Cognitive Science), at the School of Medicine (Neurosciences, Psychiatry), at the Scripps Institution of Oceanography, the VA Medical Center, The Salk Institute, Scripps [unreadable] Research Institute and the Burnham Institute. The training faculty has considerable expertise in the anatomy, biochemistry, molecular biology, genetics, physiology, and modeling of systems of neurons. Most of the students funded by this grant are from Neurobiology, Computational Neurobiology, and the Neurosciences Graduate Programs. All three programs attract a very high caliber of graduate students. Previous graduates from this program have been very successful as academic and medical research scientists. The training program uses an array of graduate courses, seminars, journal clubs, laboratory rotations, and directed laboratory research experiences to prepare them for selecting a mentor and a research project. This training grant has been used to enhance the training program by supporting a seminar series by visiting neuroscientists selected by the students; by participating in an annual Neurosciences Retreat, for the trainees to present their research results and plans; and by having a yearly course, with lectures, readings, and discussions of a particular topic chosen by the trainees and directed by 3-5 faculty members. The training program emphasizes the development of research expertise in basic areas of modern integrative neurobiology, and the trainees are encouraged to develop broad interests in this rapidly growing field. [unreadable] [unreadable] [unreadable]

Hormones have strong effects on behavior. The sex hormones estrogen and testosterone, for example, are responsible for major differences in the behaviors of men and women. The Kristan laboratory studies how hormones initiate and control behavior. To get a precise understanding of these mechanisms, they work with an animal, the medicinal leech, which produces a variety of complex behaviors with a relatively simple nervous system. Studying the control of swimming, crawling, and feeding, they have elucidated how leeches produce behaviors and choose among them. Understanding gained from these studies has informed experiments on more complex animals, including humans, which employ the same mechanisms to decide among possible behavioral responses. The Kristan group will use the same electrophysiological, pharmacological, and behavioral techniques that have proven so successful for them in understanding behavioral choice to study the cellular basis for behavioral modulation by hormones. They will determine how an analog of vasopressin causes courtship, copulation, and egg deposition in medicinal leeches. Vasopressin is a peptide hormone that is broadly distributed across the animal kingdom. All animals, from snails to humans, use vasopressin to induce and modify reproductive and parenting behaviors. Treating leeches with a homologue of vasopression reliably elicits leech reproductive behavior. The Kristan group will characterize activity in the nervous system before and during conopressin treatment. Their studies will show how this hormone causes individually identified neurons to change their activity and induces the animal to produce the constellation of behaviors that are essential for successful reproduction and, thereby, the continuation of the species. This project, from its beginning, has been conducted primarily by undergraduates, and a major component of the project will continue to give these students an opportunity to take part in cutting-edge research.

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

Dopamine (DA) is an important and universal modulator of motor control, but neuroscientists have yet to determine precisely how DA-containing neurons and their targeted circuitry choreograph specific locomotor programs. This has been an especially daunting task in studying the control and initiation of locomotion in higher vertebrate systems. Related to this issue of locomotor regulation is the idea of decision-making, and how one form of locomotion is selected instead of another, for example, the choice between crawling vs. swimming. This collaborative research project is addressing such important questions at the level of single identified neurons; often at times while the intact animal is behaving. The simpler nervous system of the leech, Hirudo medicinalis, was selected for study because it contains relatively large and physiologically accessible neurons and a hierarchical circuit organization, thus facilitating studies of locomotion, body movement and descending control. Specifically, the collaborative research team is characterizing constituents of the pattern-generating network underlying crawling-related behavior, and determining how DA changes the properties of neurons to facilitate their participation in crawling. This approach will lead naturally to an understanding of the neuronal bases of decision-making, because it has been found that whenever DA triggers crawling then swimming is inhibited. The collaborative research team will use a variety of behavioral, electrophysiological, and anatomical methods to study how DA promotes crawling behavior. They will also image neuronal circuits influenced by DA using a state-of-the-art voltage sensitive dye imaging system. Many of the experiments being conducted involve graduate and undergraduate students. Other experiments, with minor modification, incorporate the participation of younger K-12 students. The collaboration with Dr. Crisp at St. Olaf College, an undergraduate-only research institution, provides an additional and valuable exchange of undergraduate training and mentoring opportunities. The projects and technological components of the proposal are inherently integrative, spanning disciplines from Animal Behavior to Cell Biology/Neurobiology, Computational Neuroscience, Physics, and Engineering.

Vasopressin and oxytocin are among the most highly conserved molecules in all of biology, both in their molecular structure and in their functions. They are small peptide hormones that control reproductive behaviors (e.g., courting, copulation, infant care, pair-bonding) in a broad range of animals from snails and worms to rodents and humans. However, hormones do not by themselves produce these important behaviors. Instead, they act on the nervous system, changing its activity and as a result, changing behavior. This study will use a relatively simple model organism, the European medicinal leech, to study how a neuronal circuit is activated by the animal's version of oxytocin and produces courtship behaviors. The investigators will use modern molecular techniques (mass spectroscopy) to identify the leech version of oxytocin, and will use cutting-edge electrical and optical techniques to characterize the set of neurons that are activated by the hormone. Based upon their previous findings, they expect to find that the leech, like mammals including humans, uses the oxytocin analog both as a neurotransmitter between specific neurons and as a hormone that modifies the properties of all the neurons. They will be able to trace these effects from the molecular mechanisms to the behavior produced. This characterization of the leech system will be the first complete description of the circuitry underlying any reproductive behavior, and it will show how the same molecule can function both to activate behaviors and help to produce them. The results will serve as a springboard to inspire similar characterizations of neural networks for reproductive behaviors in more complicated animals, including mammals. This project will offer unique training opportunities for students who will participate in every phase of this research project.