Donald H. Edwards, Ph.D. - US grants

While neural command systems are thought to play an important role in organizing the behavior of animals, little is known about the natural excitatory inputs to most command systems or about inhibitory interations among them. The long-term objectives of this research are (i) to describe how identified sensory neurons in crayfish excite identifiable command neurons (CNs) that release abdominal movements and (ii) to describe how inhibitory interactions among command systems prevent the simultaneous release of mutually exclusive behavioral responses. Descriptions of these phenomena should provide insights into the neural bases of behavioral choice in this and other animals. The sensory excitation is provided by a set of central nervous photoreceptors, the caudal photoreceptor neurons (CPRs), which excite command interneurons in the rostral nervous system. These CNs project caudally to release backware walking and abdominal postural movements and to inhibit other command systems, including those for escape. Escape CNs inhibit the abdominal postural motoneurons and may inhibit abdominal postural CNs. These inhibitory interactions among central command systems appear to allow the animal to do one thing at a time. The proximate goals of this application are: (1) to identify abdominal postural CNs in the rostral CNS that are excited by the CPR neurons; (2) to determine the anatomical relationship between the CPR terminals and the input regions of the command cells; (3) to record the spike and synaptic responses of those command cells to CPR spike trains; (4) to describe the interactions between CPR and other afferent inputs to the CNs; and (5) to describe the inhibitory relations between abdominal postural CNs and escape command cells. Extracellular recording and stimulating techniques will be used to identify the axons of abdominal postural command interneurons within interganglionic connectives. Responses of CNs to electrical CPR stimulation will be recorded alone and during sensory stimulation that normally excites or inhibits those neurons. Responding CNs will be stained and their effects on postural motoneurons recorded. Both CPRs and the CNs they excite will be traced electrophysiologically and anatomically to their site of interaction in the rostral CNS. The command neurons will be penetrated and their synaptic responses to CPR activity recorded. Synaptic inputs from other afferents and command interneurons will also be recorded.

No abstract provided.

This research will investigate the mechanisms of synaptic integration in the lateral giant (LG) command neurons that trigger the escape tailflip of both developing and adult crayfish. Two questions will be addressed. The first asks how the LG's responses to mechanosensory inputs depend on the responses of tonic and phasic mechanosensory interneurons (MSIs) to tactile stimuli, the pattern of synaptic contact of individual MSIs on LG, the morphology of LG, and the passive and active membrane properties of LG. This question will be addressed by recording the responses of two identified MSIs, one phasic and one tonic, to mechanosensory stimuli that excite LG, by identifying possible MSI contact sites on LG and by recording LG's responses to them individually and in various patterns. The structural, biophysical and synaptic response data will be used to reconstruct the integrative properties of LG in a quantitative model that will acconut for the cell's responses to its normal patterns of input. The second question asks how the integrative properties of LG change during the animal's growth from post-natal fry to adult, and whether those changes and accompanying changes in synaptic inputs to LG can account for changes in tailflip behavior. We will analyse LG integrative properties in 2 cm, 6 cm and 12 cm animals in the manner described above, and develop quantitative models of them to determine how growth - related changes in cell shape, membrane properties and synaptic inputs should affect LG. We will also determine what changes occur in the number and pattern of inputs LG receives from identified MSIs and from the set of mechanosensory afferents. This study should identify the compensatory mechanisms that allow the labile disynaptic pathway to LG to remain viable during growth while the non- labile monosynaptic pathway fails. Since neuronal growth occurs in all nervous systems, both the changes in integrative properties during growth that we observe and the mechanisms that compensate for those changes may prove to be common features of developing nervous systems.

biomedical equipment resource; biomedical equipment purchase;

Social relationships among animals influence their behavior, and do so by affecting the neural circuits and responses that control behavior. A primary link between social status and behavior occurs through the release of neuromodulators that have both widespread and targeted effects within the nervous system. One neuromodulator, serotonin, has been implicated in the expression of social ordering behaviors in a wide range of vertebrates and invertebrates, including primates, rodents, fish and crustaceans. The neural mechanisms that link serotonin's effects to the expression of dominance status have been studied most thoroughly in crustaceans, particularly lobsters and crayfish. During the current grant period the social dominance status of a crayfish was found to predict the effect of the neuromodulator serotonin on the neural circuit for the taiflip behavior. Serotonin enhances the response of the Lateral Giant (LG) tailflip command neuron to its normal mechanosensory input in isolated crayfish and in socially dominant crayfish, and inhibits the cell in subordinate animals. The differences result from corresponding differences in the population of serotonin receptors. The present proposal continues this research by determining the sources of serotonin that modulate LG in freely behaving animals, and by asking where and how serotonin has its different effects on LG in the animals of different social status. Blood levels of serotonin in freely behaving crayfish will be measured during fights, and the responses of LG to serotonin, serotonin agonists and antagonists will be measured with standard electrophysiological techniques.

Electroreception is a special sensory ability of some animals to detect extremely small electrical fields in the natural environment. It was discovered in aquatic vertebrates less than 40 years ago, and this special sense has since been discovered in mammals. However, it has never been found in any invertebrate. This project uses physiological and behavioral techniques to design tests for the presence of electrosensory capabilities in crustaceans. The SGER is an appropriate mechanism for this unique opportunity because the experiments are brief and straightforward, and while the risk is conceptual that the PI's might not find electroreception, the potential impact of the discovery is very high. Such a discovery would have immediate importance not only in opening an entirely new field for sensory science, but also in offering new concepts and approaches for neuroscience in general, for evolutionary biology and potentially for bioengineering.

The East Coast Nerve Net is a small conference that encourages interaction among graduate students, post-doctoral scientists and faculty in an intensive weekend. The conference utilizes the facilities of the Marine Biological Laboratory in Woods Hole, Massachusetts, an outstanding institution for biological research, which provides an informal setting. The presentations emphasize participation by young scientists who have not yet presented their work at major national or international meetings. This award helps defray participant costs to encourage a few undergraduate students of under-represented groups to present their work in talk or poster form. This conference participation will allow a few outstanding undergraduates to gain experience and information from their peers and mentors that will be important for their career development.

DESCRIPTION (provided by applicant): The long-term objectives of this proposal are to understand the neural bases for the changes in behavior that accompany changes in an animal's social status. Crayfish are an appropriate model because the differences in the behavior of dominant, subordinate and single (isolated) animals are well described, and the neural circuits that mediate relevant patterns of behavior, such as escape, have been well studied. The Lateral Giant (LG) interneuron in the escape circuit serves as a command neuron for the behavior. LG's excitability can be modulated by serotonin (5HT), but the sign of this modulation depends on the social status of the animal: 5HT facilitates LG's response in social dominant animals and inhibits it in social subordinates. The effect of serotonin in social isolates depends on the manner in which it is applied, whether fast or slow, at higher or lower concentrations, and for shorter or longer periods. These last effects, which are of clear clinical relevance, are hypothesized to result from activation of competing second messenger pathways through separate serotonin receptors, whereas the effects of social status may result from changes in the population of available receptors in the different animals. Accordingly, the first aim of the proposal is to describe the set of serotonin receptors that modulate LG's response by cloning, sequencing and expressing crayfish CNS serotonin receptors. Antibodies to those receptors will then reveal their location, and the discovery of selective agonists and antagonists will enable their different effects to be determined, cAMP mediates one effect of 5HT on LG; other 2nd messengers pathways will be identified and their interactions studied through the use of specific agonists and antagonists that can be either applied extracellularly or injected into the giant neuron. One effect of 5HT, long-term facilitation, is also produced by repeated bouts of sensory nerve stimulation; this pattern of stimulation is also known to evoke 5HT release. Another aim will be to determine whether release of 5HT mediates stimulus-induced long-term facilitation. Finally, the afferents to LG are electrically coupled; the final aim will be to test whether this coupling forms a lateral excitatory network among the afferents to select and amplify phasic inputs to LG. This will be studied with standard electrophyiological techniques.

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 (witness Kosovo). 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.

DESCRIPTION(Adapted from applicant's abstract): Serotonin is a neurotransmitter and neuromodulator used in the control of aggression, movement, and gut motility in vertebrates and invertebrates. In the past few years, serotonin has been found to be "borrowed" rather than synthesized by some central and peripheral neurons in vertebrates. Recent results indicate that a set of 60 neurons that innervate the hindgut of crayfish employ serotonin as a neurotransmitter. Instead of synthesizing serotonin, however, these hindgut neurons (HGNs) appear to "borrow" serotonin by taking it up from external sources. These uptake mechanisms appear to be under the control of exteroceptive and proprioceptive sensory input. The overall goal of this research is to investigate the mechanisms that underlie this "borrowed transmitter" phenomenon in craylish, and to determine how it contributes to the control of serotonergic modulation in the crayfish. Specifically, the proposed research will determine quantitatively whether serotonin is preferentially borrowed or synthesized by the HGNs. The mechanisms that govern borrowing, including those that controi the supply of serotonin made available in the vicinity of the HGNs by external sources, and those that control serotonin uptake and release by the HGNs, will be investigated. The use to which serotonin is put by the HGNs will be determined, as will the effects of serotonin uptake on other adjacent and well-studied serotonergic systems, including those that modulate posture and escape.

Resistance reflexes help an animal to counter external forces that would move its limbs, thus they help maintain a stable posture. During voluntary movement, those same resistance reflexes must be disabled or reversed to allow the movement to occur. This study asks how the resistance reflexes are organized to allow an animal to maintain a static posture, and how they are disabled and transformed into assistance reflexes at the onset of voluntary movements. While these questions have been approached in vertebrates, insects and crustaceans, answers are far from complete. The questions in the current study will be addressed in the crayfish because relevant circuitry for leg movements has been described in terms of a set of identified sensory neurons, interneurons and motor neurons, and their synaptic interactions.

The investigators will use three approaches: (i) in vivo motion analysis and nerve recordings; (ii) a hybrid in vitro preparation/neuromechanical model; and (iii) a neuromechanical simulation of the crayfish body and nervous system. This approach will reveal how the animal controls its reflexes to help stabilize posture and assist voluntary movements underwater and on land, where the set of external forces are very different. The mechanisms discovered here are likely to be used by other legged animals. The approach employs two new tools developed by the principal investigator's laboratory, the AnimatLab neuromechanical simulation software and the hybrid neuromechanical interface that links a model to the nervous system of the experimental preparation. AnimatLab is freely available at www.animatlab.com, and the hybrid interface will be made into a commercial-grade electronic instrument. The research will provide training in neurophysiology, behavioral analysis and computational neuroscience to undergraduate and graduate students who include members of underrepresented populations. Those students will participate in helping to teach relevant portions of the summer B.R.A.I.N. course at Georgia State University for outstanding undergraduates from around the country.