Janis Weeks - US grants

We plan to continue our investigation of the causes and functional consequences of dendritic regression in proleg motoneurons (MNs) in the tobacco hornworm, Manduca sexta, with these objectives: (1) to characterize anatomical and electrophysical changes in the monosynaptic connections between planta hair afferent neurons and the proleg retractor MNs during the larval-pupal transformation. This is the time during which the MNs undergo their hormonally-mediated regression and the proleg withdrawal reflex disappears. These studies will involve conventional neuronal staining techniques in combination with intracellular neuronal recordings to determine the number and strength of afferent-to-MN synapses. Possible pre- and postsynaptic causes for the weakening of the afferent-to-MN reflex will be sought. (2) To test possible causal relationships between afferent input to the MNs and MN structure, both during larval development and during the larval-pupal transformation. These experiments will involve surgical ablation of prolegs during larval life, and hormonal treatments to produce heterochronic mosaics with mixed larval and pupal attributes. Anatomical and electrophysiological studies will examine the possible causal roles of afferent synaptic input in the attainment of the larval morphology of the MN, and in the subsequent regression of the larval arbor during pupal development. (3) The final objective is to expand these studies to include interneurons (INs). Excitatory and inhibitory INs which provide monosynaptic input to the proleg MNs will be sought, and the status of these synapses during the larval-pupal period will be followed anatomically and electrophysiologically.

This action is to recommend Dr. Janis Weeks for the Presidential Young Investigator Award. Her specialty is the electrophysiology of invertebrate systems. She has already contributed a great deal to the understanding of the leech nervous system and how it produces the swim rhythm. Her work on the characterization of moth eclosion motor patterns established her as one of the premier neurobiologists in the business of explaining pattern generation in neural terms. She has also made major contributions regarding how specific hormones control motor programs in invertebrates. This body of work has lead her into the area of hormones and how they effect the developing nervous system and behavior. Since most of our detailed information about the human nervous system comes from earlier studies first done on invertebrate preparations it is wise to support the best and the brightest of these scientific types. Dr. Weeks is of that category.

The objective of this research are to determine how hormones cause structural changes in neurons during development, how such structural changes lead to changes in synaptic connections, and how changes in synaptic connections contribute to the assembly and dismantling of neuronal circuits underlying behavior. During metamorphosis of the moth, Manduca sexta ecdysteroid hormones control metamorphic events in the nervous system including neurogenesis, programmed neuron death, and the growth and regression of neuronal arbors. These parallel the effects of gonadal sex steroids on the developing vertebrate nervous system. During larval-pupal development of Manduca, ecdysteroids cause the dendrites of identified motorneurons (MNs) innervating abdominal proleg muscles to regress severely. Concomitantly, the proleg behaviors disappear. The hypothesis that regression of proleg MN dendrites causes the loss of synaptic inputs, thereby removing the MNs from behavioral circuits, will be tested during electrophysiological and anatomical studies of monosynaptic connections from afferent neurons and interneurons to the proleg MNs. Developmental changes in synaptic connections will be related to behavioral changes. Other MNs that do not regress, but which nonetheless show alterations in synaptic drive during this time, will be studied for comparison. Some experiments will use heterochromic mosaics, in which some neurons are retained in the larval stage while their synaptic partners become pupal. After pupation, some regressed proleg MNs die; the survivors are stimulated by ecdysteroids to regrow their dendritic arbors during adult development, and they take on new behavioral roles. Changes in synaptic inputs to these growing MNs will be studied, to compare with the findings during their regression. Finally, proleg MN regression and death can be induced by directly infusing ecdysteroids into the blood of larvae. Hormone infusions will be paired with treatment with inhibitors of DNA, mRNA or proteins synthesis, to determine the necessity of these synthetic events. One hypothesis to be tested is that MN regression and death are controlled by sequential critical periods of hormone action. In summary, the proposed experiments employ a range of approaches to gain a better understanding of how hormones influence neurons and neuronal circuits. These findings ought to be relevant to all animals in which hormones influence the developing nervous system.

The applicant, Janis C. Weeks, is an associate professor (without tenure) at the Institute of Neuroscience at the University of Oregon. Her long- term career goals are to understand how hormonal effects on the structure and synaptic connectivity of identified neurons lead to changes in behavior. During metamorphosis of the moth, Manduca sexta, ecdysteroid hormones control metamorphic events in the nervous system including programmed neuron death, and the growth and regression of neuronal arbors. These morphological changes form a substrate for the massive behavioral changes that accompany the transitions between life stages. The proposed experiments address several issues related to how ecdysteroid-induced regression and/or growth of the dendritic arbors of identified motoneurons during metamorphosis affects their behaviorally-relevant synaptic inputs from identified sensory neurons and interneurons. Electrophysiological and anatomical (light and electron microscopic) techniques will be used. In addition, inhibitors of DNA, RNA, and protein synthesis will be tested for their ability to block the ecdysteroid-triggered dendritic regression, and programmed death, of identified motoneurons. This research program is in a rapidly expanding phase, and several of the projects are beginning to require a great deal of direction. It is essential, therefore, that Dr. Weeks spend as much time as possible in the laboratory, both to carry out experiments and to guide the research of her graduate student and postdoctoral collaborators. Dr. Weeks now carries a burdensome teaching load of undergraduate and graduate courses that detracts significantly from her time spent on research. If awarded an RCDA grant, she would be relieved of all teaching responsibilities at the University of Oregon except a yearly graduate course in her research area, and of all committee and administrative duties. This would allow her to spend essentially fulltime on research activities. In addition, some of the extra research time provided by an RCDA award would be used to initiate pilot studies in collaboration with Dr. Richard B. Levine at the University of Arizona, to test the feasibility of addressing some questions of hormone action on identified Manduca neurons in cell culture. These studies would not otherwise be undertaken. In summary, an RCDA award would significantly enhance the applicant's development as an independent researcher by allowing her to devote essentially full-time to her expanding research program during a critical phase of her career. Furthermore, the opportunity would be provided to pursue a potentially very powerful new research direction.

minority institution research support; secondary schools;

This research will investigate, at the level of an identified neuron, extrinsic and intrinsic factors that regulate neuronal phenotype in a steroid-sensitive system. In the hawkmoth, Manduca sexta, steroid hormones (ecdysteroids) orchestrate the postembryonic reorganization of the nervous system during metamorphosis. Steroid hormones likewise regulate neurodevelopmental events in vertebrates. Insect and vertebrate steroid receptors belong to the same superfamily of DNA-binding transcription factors, suggesting that molecular genetic mechanisms may be shared. Identification of fundamental mechanisms by which steroid hormones and other factors influence neuronal phenotype should assist in developing clinical strategies to prevent or treat birth defects and neurological disorders in humans, especially those involving endocrine malfunctions. The proposed experiments will focus on a larval motor neuron in Manduca, designated APR. APR homologs occur in each abdominal segment, where they innervate a proleg retractor muscle. An elevation of ecdysteroids at pupation causes APR's dendrites to regress dramatically, followed by the programmed death of APRs in a subset of segments. APR's target muscle also degenerates in a segment-specific pattern, but the pattern does not match that of the APRs. APRs that survive pupation are respecified for new functions in the pupal stage. This unique segment-specific pattern of APR fates provides an exceptional opportunity to investigate how extrinsic and intrinsic factors cause segmentally homologous neurons to express different phenotypes in response to the same hormonal cue. SPECIFIC AIM 1: To test how extrinsic factors (interactions with muscles, sensory neurons and interneurons) and intrinsic factors (ecdysteroid receptors; EcRs) contribute to the segment-specific fates of APRs in vivo. We will investigate the effects of manipulating the APRs' interactions with target muscles and afferent inputs, and determine whether developmental changes in interneuronal inputs to APRs vary by segment. The segmental pattern of EcR expression in APRs will also be determined. The working hypothesis is that the segment- specific pattern of APR death results from the activation, via EcRs, of intrinsic developmental programs that do not depend on interactions with other cells. SPECIFIC AIM 2: To use cell culture to test the hypothesis that dendritic regression and segment- specific death of APRs results from the activation via EcRs, of intrinsic developmental programs that do not depend on interactions with other cells. Fluorescently labelled APRs will be placed in low density cell culture and treated with physiological levels of ecdysteroids. EcR expression in cultured APRs will also be determined. If ecdysteroids evoke regression and/or death, we will begin to investigate the underlying mechanisms.

9420796 Weeks Hormones often exert dramatic effects on animal behavior, yet the mechanisms by which they act on the nervous system to alter behavior are not yet well understood, The identification of mechanisms for hormone-nervous system interactions is facilitated by studying animals with simple and accessible nervous systems. In this project, a group led by Dr. Weeks will utilize the hawkmoth, Manduca sexta, to study profound neural and behavioral changes in response to developmental alterations in steroid and peptide hormone levels. A specific behavior during the initial phase of metamorphosis in Manduca. pre-ecdysis behavior, is performed to loosen the old cuticle before it is shed at each molt. The performance of this behavior is attenuated at specific times during development. This work will correlate changes in hormones with functional changes in specific identified brain cells. The results of this research will be relevant to understanding neural mechanisms of hormonally-mediated behavioral changes in a wide variety of living organisms. ***