Edward Kravitz - US grants

A long range objective of our studies is to understand how neurohormonal systems work. Our studies have concentrated on a lobster system involving the amines serotonin and octopamine, and the peptide proctolin. When serotonin and octopamine are injected into freely moving lobsters, they cause animals to assume sustained opposite postures: serotonin causes flexion and octopamine extension of the abdomen, claws and walking legs. the postures result from amines directing the readout of central motor programs for flexion (serotonin) and extension (octopamine). At the same time the amines and the peptide, proctolin, act on peripheral exoskeletal muscles to prime them to respond more vigorously. We have located the major sites of storage and release of these substances in lobsters and have found individual neurons likely to contain serotonin that may play a role in the generation of the postures. This application proposes to extend these studies by: first, using immunohistochemical methods, locating cells containing proctolin and octopamine (verified by chemical assays) then examining the neurohormone-containing cells to try to learn how they are activated, and the consequences of their activation on aspects of lobster behavior; second, examining further the mechanisms that underly the long-lasting nerohormone-induced actions on exoskeletal muscles by attempting to develop a skeletal muscle membrane preparation for detailed physiological anc biochemical studies, and also to continue studies with intact preparations on the possible involvement of cyclic nucleotides in these processes; third, in preliminary behavioral experiments we will explore the consequences of destroying amine neurons on aspects of lobster behavior. In a fourth set of experiments we will use lower vertebrane preparations to see if we can (1) identify unique amine-containing neurons and (2) develop methods for examining specific synaptic contacts in the spinal cord using quantitative physiological methods. Amine neuron systems in man have been implicated in disorders and diseases of the nervous system like Parkinson's Disease, depression and schizophrenia and in many important normal processes like sleep, attention, initiation of movement and learning. Our belief is that detailed knowledge of how an amine-associated system (like our lobster system) works, will generate useful ideas and experimental avenues of approach toward understanding the enigmatic and much more complicated amine-neuron systems of man and higher vertebrates.

The project consists of closely related studies, carried out by six independent investigators and their coworkers, examining the generation, modulation and developmental regulation of identified circuits involving amines and peptides, studying their functions at a cellular and systems level, and asking how these substances influence behavior. Seven Projects and five Cores make up this Program Project application. The titles and responsible investigators are: Project 1. Developmental and hormonal modulation of synapses (EA Kravitz, PI); Project 2. Studies of multi- transmitter modulatory neurons in the stomatogastric system (R Harris- Warrick, PI); Project 3. The roles of identified serotonin and octopamine neurons (EA Kravitz, PI); Project 4. Biochemical and physiological studies of cGMP metabolism (MF Goy, PI); Project 5. Development of amine neurons and their targets (BS Beltz, PI); Project 6. Molecular genetic studies relating to lobster neurohormonal substances (H Potter, PI); Project 7. Chemical signals regulating social behavior (J Atema, PI); Core A. The lobster rearing facility (BS Beltz, PI); Core B. Summer rental at the Marine Biological Laboratory (MF Goy, PI); Core C. Photography (EA Kravitz, PI); Core D. Shop (H Potter, PI); Core E. Administration (EA Kravitz, PI). Amines and peptides are important compounds in human nervous system function. They have been implicated in diseases and disorders of the nervous system as diverse as Parkinson's Disease, schizophrenia, sleep disorders, chronic pain, abnormal violent behavior and affective disorders. Yet the details of how these substances function in influence these and other important processes remain elusive. Together the members of this Program Project present a coordinated and broadly based program of exploration of the functions of amines and peptides in a single species, the lobster. The studies range from molecular biology to behavior, and that wide a program of investigation would be difficult for any member of the group to pursue alone. The combined effort represented by the Program Project commits its members to working and thinking together on important issues surrounding the theme of amine and peptide neuron function. Hopefully the results of this coordinated effort will generate valuable data and useful models of how these important molecules function. Therein lies the strength of this application, and the rationale for this Program Project.

9728551 Kravitz Serotonin has been linked to aggression in a wide and diverse range of species, including man. In vertebrates, lowered levels of this amine, possibly coupled with elevated levels of norepinephrine, have been linked to a particular kind of impulsive violence. While great interest is centered on this research, it is difficult in vertebrate systems, because of the large number of neuronal elements involved, to link amines and aggression at the level of single neurons and the precise changes taking place in the nervous system accompanying violent behavior. In invertebrate systems, with their limited numbers of large identifiable neurons, the analysis of complex behavioral processes, like aggression, can be brought to the level of the precise cells thought to be essential for the behavior. This application continues our studies exploring the function of identified single nerve cells from the lobster central nervous system (cns) that contain and utilize the amines serotonin and octopamine (the invertebrate equivalent of norepinephrine). We believe that these two amines have opposing actions on at least the postural component important in agonistic (fighting) behavior in lobsters. The serotonin cells that are the focus of our studies are neurosecretory cells. These cells release serotonin at two sites: (i) within the central nervous system, where it influences pathways having to do with posture and escape; and (ii) directly into the haemolymph (blood) of the lobster, where the amine changes the ways that muscles and sensory neurons function for prolonged periods of time. Within the cns, much in the same way that dimmer switches work on electrical circuits, the serotonin cells function as "gain-setters" that make particular pathways function better or worse for lengthy periods of time. The present studies will continue our investig ations of these interesting neurons, asking how they are activated, and will begin studies aimed at asking whether these serotonin-containing neurons show any changes in functioning as a long-term consequence of changes in social status. With the octopamine neurosecretory cells we plan studies aimed at defining their physiological roles, learning how they are activated, asking how and where they interact with the corresponding sets of serotonin-containing neurons, and asking whether a subset of these cells that selectively innervate the claws, serve any direct role in the switch to subordinate behavior seen when animals lose one of their large claws. The emphasis in all of our studies is on understanding, at a neuronal level, the short-and long-term consequences of social interactions between animals. With its well studied biochemistry, physiology, anatomy and behavior, the lobster model we are examining, offers many advantages in addressing such issues.

This proposal has the overall goal of understanding the roles served by neurohormones in a complex behavior, like aggression. It involves investigators from 3 universities: E. A. Kravitz (Harvard Medical School), D. H. Edwards (Georgia State University), and B. S. Beltz (Wellesley College) who use crustacean model systems (lobsters, crayfish) for their interactive studies. Crustacean models allow the analysis of behavior to be brought to the level of the individual nerve cells involved, an advantage not readily available in higher forms. The studies focus on serotonin, an amine derived from the common amino acid tryptophan, which has been implicated in aggression in all animal species. They explore the effects on fighting behavior of raising and lowering serotonin levels, ask whether neurons utilizing this amine function differently in dominant and subordinate animals, and address the question of whether these neurons change their properties over individual molt cycles (the annual shedding of the cuticle) along with behavioral changes that are seen.
Aggression is a universal feature of the behavior of social animals, necessary for access to shelter, food and mates. Among humans, the consequences of its abuse are well known. Despite the importance of this behavior, however, little is known of its neural mechanisms. Models, like the crustacean systems used here, offer the possibility of highly detailed analyses of this complex behavior, thereby yielding valuable insights into its underlying neural roots.

A common behavior among animals is aggression, which can occur during competition for food, mates, or other limited resources. As a consequence of winning or losing fights with other individuals in the same social group, subsequent behavioral patterns change, as winners can dominate losers. A biochemical compound, the amine serotonin, is known to be important in aggression, but other hormonal substances also have been implicated including peptides and steroids, which may interact with amines. The lobster is a social crustacean that shows complex stereotyped behavior, and offers the opportunity to examine the hormonal modulation of behavior at the level of single identifiable cells in the brain. Specific sets of cells containing amines have been mapped, and a group of neurons containing stress-related peptides has been identified that interact with the amine neuron sets. This project examines the cellular networks of the lobster neurons containing and responsive to serotonin and certain peptides and steroids. Electrophysiological recordings and morphological analyses will reveal how the systems are activated, and detail the synaptic interactions and circuitry involved.
Results will be important for behavioral neuroscience as well as neuroendocrinology, in clarifying cellular mechanisms underlying the hormonal regulation of aggression, which is an important behavior in humans as well as animals. This project also will have an impact through its excellent integration of education and training with research, including worldwide collaborations and outreach to under-represented groups.

DESCRIPTION (provided by applicant): Aggression is a nearly universal feature of the behavior of social animals. In the wild, it is regularly used for access to food and shelter, for protection from predation and for selection of mates. Despite its importance, little is known of the neural mechanisms that underlie the behavior. In almost all species of animals in which it has been looked for, aggression and/or territorial behavior has been found. It is not surprising, therefore, to find that fruit flies also show aggression and territorial behavior. The existence of these behaviors in Drosophila species is not widely known, however, with the exception of the difficult to study Hawaiian species. With powerful genetic and molecular methods available to explore fundamental biological processes, Drosophila melanogaster offers unique opportunities for studies aimed at understanding how a complex behavior like aggression is assembled and activated within, and impacts upon the nervous system. The goals of these studies are to perform a quantitative analysis of fighting behavior in Drosophila melanogaster and to use the analysis to examine the consequences of specific chemical ablations and neuronal mutations on the behavior. There are two Specific Aims of these studies. In the First Aim we plan to: (1) establish conditions under which fighting is a robust phenomenon; (2) use these experimental conditions to videotape and analyze fights in order to construct an ethogram; (3) attempt to define a simplified scoring procedure for fly fights; (4) ask whether there is a long-term component to winning and losing fights in flies. In the Second Aim we plan to: (1) change the levels of amines like serotonin, octopamine and dopamine using chemical and mutant approaches and observe the effects on fighting behavior; (2) examine the fighting behavior of various learning mutants in flies; and (3) abolish mushroom bodies in the brains of flies, which are known to be important in learning and memory, using chemical and transgenic approaches and ask whether decision making ability or the short- and long-term consequences of winning or losing fights are altered. The human health issues these studies relate to are violence, aggression, drug abuse and mental health.

[unreadable] DESCRIPTION (provided by applicant): Aggression is a nearly universal feature of the behavior of social animals. In the wild, it is used for access to food and shelter, for protection from predation and for selection of mates. Despite its importance, little is known of the neural mechanisms that underlie the behavior. In this proposal a genetic approach will be taken to the examination of aggression using the fruit fly, Drosophila melanogaster, as the experimental model. With the genome sequenced, with a wealth of powerful genetic methods available, and with the recognition that similar genes are used in similar ways in all species of animals, including humans, studies with fruit flies should yield important information towards understanding this complex pattern of behavior. The long-term goals of the present studies are to identify genes and molecules, subtypes of neurons, and neuronal pathways important in aggression in fruit flies. Towards these goals, this application has four Specific Aims. I. Behavioral studies will address the questions: (1) Do females fight and can their behavior be analyzed; and (2) Are there long- and/or short-term memories of social status as a consequence of winning or losing fights. II. Amines like serotonin have been implicated in aggression in most species of animals. Mutant fly lines will be generated in which amine neuron function can be turned off and on in the brain by small changes in temperature. The powerful GAL4/UAS system will be used for this purpose, and we will selectively modify the function of serotonin, dopamine and octopamine neurons to observe the effects on fighting behavior. III. Assuming that as in most species, there will be a memory of social status: (1) fighting behavior will be examined in flies in which mushroom bodies (brain areas important in memory in flies) have been chemically ablated; (2) fighting behavior will be examined in learning mutant fly lines; and (3) GAL4 lines will be used to examine the effects on fighting behavior of reversibly ablating subsets of mushroom body neurons. IV. Parkinson's and Alzheimer's/Frontotemporal Dementia disease models have been generated in flies by creating transgenic animals with proteins known to be involved in these diseases in humans. One phenotype associated with these disorders is enhanced aggression. The fly models will be examined to see if they too show enhanced aggression. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Aggression is an innate behavior that is a nearly universal feature of the social behavior of animals. In the wild, it is used for access to food and shelter, for protection from predation and for selection of mates. Despite its importance little is known of the neural mechanisms that underlie the behavior. Over the past decade, we have developed a Drosophila melanogaster model of aggression. In same sex pairing of male and female flies, these animals will compete over resources. Males develop hierarchical relationships while females do not, and learning and memory take place during the male fights. A single gene, fruitless, specifies both how flies fight and who they court. Amines have been shown to be important in aggression in all species of animals examined thus far. Fruit flies are no exception. These studies focus on two major amines found in the fruit fly nervous system: octopamine (OA), the phenol analogue of norepinephrine, is the major amine synthesized from tyrosine;and serotonin (5HT), is the major amine derived from tryptophan. There are approximately 100 OA and 5HT neurons in the Drosophila central nervous system. Of the total OA pool, we have identified a small group of 3 or 4 neurons, that co-express the amine and the male protein forms of Fruitless (FruM/OA neurons). These appear to be involved in the decision made by male flies between courtship and aggression. Here we propose to use state of the art genetic methods and a combinatorial method to unravel the circuitry concerned with the FruM/OA neurons, from sensory input through to behavioral output. 5HT serve roles in aggression (not in initiating fights, but in bringing fights to higher intensity levels), courtship behavior, balance and feeding behavior in flies. We ask here whether we can identify the specific 5HT neurons involved in these behaviors, and then can map the circuitry involving the neuron or neurons concerned with aggression. The project has the following Specific Aims: Aim 1: To generate a data base of the morphological features and functional roles served by individual 5HT, OA and dopamine (DA) neurons. Aim 2: Selective manipulation of FruM/OA neurons and behavioral choice: can we map the circuitry concerned with these neurons. Aim 3: Selective manipulation of individual serotonergic neurons. Can we identify which of the ca. 100 5HT neurons are concerned with each of the behaviors known to be influenced by 5HT? If so, can we then map the circuitry involved with 5HT and aggression, the same way we plan to map the FruM/OA circuitry. Studies exploring the roles of amine neurons in behavior at this level of detail just are not possible with other model systems at the present time. PUBLIC HEALTH RELEVANCE: All organisms, including humans, must be capable of rapidly evaluating social situations and of selecting proper responses from a wide variety of possible behavioral choices. Such selections must be correctly made to allow the survival of organisms as individuals or as a species. How organisms make such choices and how the underlying neural circuitry involved in decision making is constructed are the themes of this application.

Aggression is an innate behavior essential for access to mates, food and shelter. Its pathological form, violence, is a serious problem in society. In both cases, aggression has biological roots that remain poorly understood. This project examines aggression in the fruit fly, Drosophila melanogaster, focusing on the learning and memory that accompanies winning and losing fights in male flies. Losing flies remember and fight differently against familiar and non-familiar opponents, but losers lose second fights against all opponents (a behaviorally conditioned "loser mentality" develops). With powerful genetic methods available that allow manipulation of genes anytime and anywhere desired in the nervous system, and with an important behavior to examine, fruit flies offer ideal experimental animals in which to combine genetic and behavioral approaches in asking the following questions: 1. How long do flies remember status, and does the training protocol influence the duration of the memory? 2. Do genes identified as serving important roles in learning and memory in simple test protocols in flies or in other systems serve similar roles in the more complex socially-mediated learning and memory accompanying aggression in this model? 3. What roles do amine transmitters like serotonin and dopamine serve in the learning and memory accompanying aggression? 4. Do changes in gene expression accompany changes in social status? In addition to the challenging scientific questions being addressed, this project offers a broad range of training opportunities. High school, undergraduate, graduate students and post-doctoral fellows continue to work on the project and will be involved at all levels. Teaching modules used in colleges have been designed around both of the PI's earlier lobster and the present fruit fly experimental systems and the videos generated in the Kravitz laboratory are being used in teaching behavioral biology by faculty around the world.

DESCRIPTION (provided by applicant): Amine-containing neurons are found in relatively small numbers in specific nuclei deep within the brainstem in essentially all vertebrate species, including humans. Despite their limited numbers, amine effects on behavior are profound as their neuronal processes ramify widely throughout the nervous system. Dysfunctioning of amine neuron systems has been implicated in the pathology of many psychiatric and neurological diseases. Included are motor system disorders like Parkinson's Disease in which subsets of the dopamine neurons selectively die, and psychiatric disorders including mood disorders, schizophrenia, attention-deficit hyperactivity disorder and drug abuse in which the amines dopamine, serotonin and norepinephrine all have been directly implicated. Arousal, reward, learning and memory, risk taking behavior, aggression and stress related behavioral and physiological responses are some of the multitude of essential human behaviors in which amine neurons have been suggested to play key roles. With likely roles in so many central aspects of human behavior, it is no wonder that a huge literature has grown up on amine neuron systems and how they function. Originally thought to be fairly homogeneous populations of neurons found in selective CNS nuclear regions, continuing study has revealed that these systems instead are very heterogeneous populations of neurons in terms of their function and their morphology, even within single brainstem nuclei. The most challenging part of understanding these systems now is to understand how they coordinate and/or organize the many behavioral processes they seem concerned with. A difficulty in addressing such questions in higher vertebrate nervous systems is that even though the numbers of amine neurons involved are relatively small compared to the total numbers of neurons in the brain, it is highly unlikely that one will find the same neuron more than once to explore its function in detail. Recently developed methods in other model systems, however, allow targeting and manipulation of single identified amine neurons. These experimental approaches are the farthest advanced in fruit flies (Drosophila melanogaster) where powerful genetic methods allow identification and manipulation of single neurons concerned with behavior. As in vertebrate systems, amines have been shown to be intimately involved in many essential aspects of behavior in fruit flies, including motivation and reward, locomotion and feeding, learning and memory, and courtship and aggression. The studies proposed here have three Specific Aims concerned with bringing the study of how amine neurons work in fruit flies to an identified single neuron level. They are: Aim 1: Use of a combinatorial genetic approach to identify small subsets of amine neurons; Aim 2: The behavioral consequences of altering the functional properties of single amine-containing neurons; and Aim 3: Analysis of the circuitry associated with single amine neurons. Studies at these levels of detail are not possible in other species at the present time.

DESCRIPTION (provided by applicant): Amine-containing neurons are found in relatively small numbers in specific nuclei deep within the brainstem in essentially all vertebrate species, including humans. Despite their limited numbers, amine effects on behavior are profound as their neuronal processes ramify widely throughout the nervous system. Dysfunctioning of amine neuron systems has been implicated in the pathology of many psychiatric and neurological diseases. Included are motor system disorders like Parkinson's Disease in which subsets of the dopamine neurons selectively die, and psychiatric disorders including mood disorders, schizophrenia, attention-deficit hyperactivity disorder and drug abuse in which the amines dopamine, serotonin and norepinephrine all have been directly implicated. Arousal, reward, learning and memory, risk taking behavior, aggression and stress related behavioral and physiological responses are some of the multitude of essential human behaviors in which amine neurons have been suggested to play key roles. With likely roles in so many central aspects of human behavior, it is no wonder that a huge literature has grown up on amine neuron systems and how they function. Originally thought to be fairly homogeneous populations of neurons found in selective CNS nuclear regions, continuing study has revealed that these systems instead are very heterogeneous populations of neurons in terms of their function and their morphology, even within single brainstem nuclei. The most challenging part of understanding these systems now is to understand how they coordinate and/or organize the many behavioral processes they seem concerned with. A difficulty in addressing such questions in higher vertebrate nervous systems is that even though the numbers of amine neurons involved are relatively small compared to the total numbers of neurons in the brain, it is highly unlikely that one will find the same neuron more than once to explore its function in detail. Recently developed methods in other model systems, however, allow targeting and manipulation of single identified amine neurons. These experimental approaches are the farthest advanced in fruit flies (Drosophila melanogaster) where powerful genetic methods allow identification and manipulation of single neurons concerned with behavior. As in vertebrate systems, amines have been shown to be intimately involved in many essential aspects of behavior in fruit flies, including motivation and reward, locomotion and feeding, learning and memory, and courtship and aggression. The studies proposed here have three Specific Aims concerned with bringing the study of how amine neurons work in fruit flies to an identified single neuron level. They are: Aim 1: Use of a combinatorial genetic approach to identify small subsets of amine neurons; Aim 2: The behavioral consequences of altering the functional properties of single amine-containing neurons; and Aim 3: Analysis of the circuitry associated with single amine neurons. Studies at these levels of detail are not possible in other species at the present time.

? DESCRIPTION (provided by applicant): Aggression is a normal innate behavior utilized for access to food, territory and mates by essentially all species of animals, including humans. Levels of display of aggression vary widely among individuals, however, and it generally is not known how much of this heterogeneity is genetic and how much is socially induced. Probably both mechanisms influence the expression of the behavior in all organisms, and the proportions of each that are involved vary widely between individuals. Unbridled aggression, in the form of violence, is a peculiarly human manifestation of this behavior, and when one adds the use of weapons capable of inflicting deadly damage to individuals and masses of individuals, it is a serious problem in society. Indeed weapons allow the least fit of individuals to become dominant protagonists in our society. In animal species, conspecifics sometimes kill opponents as well, but more commonly members of the same species engage in ritualistic stepwise-increasing-intensity-displays of fighting abilities. Winning and losing decisions can be made anywhere along the steps of such an intensity ladder. The roots of aggression are biological but there is little concrete information of how and where in the nervous system the seeds of violence are sown. In this application we propose to use a Drosophila model of aggression that we pioneered the use of in its modern form. Of all the available models for aggression, the Drosophila system offers the greatest ease and reproducibility of genetic manipulation within the nervous system down to single neuron levels. These manipulations can readily be combined with quantifiable behavioral measures in attempts to understand this complex behavior. Recently, using a novel strategy called intersectional genetics, we identified and manipulated in behaving animals single serotonin, dopamine and octopamine (fly equivalent of norepinephrine) neurons that all are involved in aggression. Thus, a single pair of serotonergic neurons found via this route, facilitat going to higher levels of aggression during fights, while a single pair of dopaminergic neurons are required to generate short term winner effects. In this application we ask a series of questions about the high-level aggression used by males to win fights. (1) What neurons and circuits are involved in going to high-intensity levels during fights, how do they work and how do they form during development? (2) What genetic or wiring differences exist in the nervous systems of the parent strain of flies and a hyper-aggressive line we generated called bullies that fight at higher intensity levels and always win fights against the parent strain? (3) Can we explain at cellular and circuit levels the learning and memory that takes place during fruit fly fights and accompanies the generation of winner and loser flies with changed aggression profiles? This application addresses the question of whether science and the study of model organisms can explain even a small part of the serious and pressing issues surrounding the root causes of human violence.