This grant will be used to acquire equipment with which to conduct neurophysiological experiments. The equipment will consist of a vibration isolation table, micromanipulators, oscilloscope, appropriate amplifiers, tape and chart recorders, and stimulator. The research conducted with this equipment will determine the precise influences circulating hormones have on a well-defined neural network. The preparation used will be the Stomatogastric Nervous System (STNS) of the spiny lobster Panulirus interruptus. The STNS controls all movements associated with the stomach. In lobsters, chewing occurs internally, in the gastric mill region of the stomach. The stomatogastric ganglion (STG) in particular produces two distinct motor patterns associated with the stomach movements. Molting in lobsters is controlled by two types of circulating hormones - a collection of steroid hormones collectively called ecdysteroids, and a peptide hormone called Molt Inhibiting Hormone (MIH). During the molt cycle, the circulating levels of MIH decrease, and this triggers a rise in ecdysteroids (Figure 1). Molting occurs once ecdysteroid levels have reached a peak and begin to decline. The lobster stops eating prior to molting and sheds the lining of the stomach during the molt. My working hypothesis thus is that MIH plays an essential role in maintaining the day-to-day functioning of the STNS. I further expect that declining levels of MIH, or increasing levels of ecdysteroid, will initially inhibit the motor patterns of the STG. The subsequent drop in ecdysteroid levels that triggers the molt will in turn elicit a novel motor pattern associated with shedding the lining of the stomach. Molting in crustaceans can be induced by removing the eyestalks of juvenile animals. The eyestalks contain the neurosecretory cells that release MIH. Eyestalk ablation leads to an increase in ecdysteroids. Because the animal itself is making the hormones, the appropriate ecdysteroids are pro duced in the proper ratios. I will use anatomical and physiological techniques to examine the effects of circulating hormones on the STNS. My specific aims will be: Aim 1: Determine the precise concentrations of circulating ecdysteroids during the molt cycle in the spiny lobster. I have already initiated a collaboration with Dr. D. Watson at the University of Alabama, Birmingham in which he will run radioimmunoassays to determine the circulating levels of ecdysteroids during induced molts. Serum samples will be withdrawn from animals during various stages of the molt cycle to determine the time-course for hormonal changes during molting. Aim 2: Examine the change in expression of modulatory neurotransmitters. A single result from an early experiment in Cancer borealis suggests that the expression of certain modulatory transmitters is downregulated during natural molts. I will use anatomical and immunocytochemical techniques to measure the expression of identified transmitter substances within the STNS in eyestalk ablated and control animals. Aim 3: Examine changes in STNS motor patterns and individual network neurons prior to and during molts. To do this, I will use a combination of intracellular and extracellular recordings to monitor rhythmic motor patterns. Preliminary work will consist of extracellular and intracellular monitoring of the STG motor patterns in molt-induced and control animals. In later experiments, I will use voltage clamp techniques to monitor changes in ionic conductances in individual neurons. Aim 4: Characterize changes in the effects of modulatory inputs to the motor pattern generating network. Previous research I conducted suggests that the effects of modulatory inputs to the STG is altered prior to a spontaneous molt. I will use bath application of identified and characterized neuromodulators as well as stimulation of individual inputs to compare effects during and between molts. The work will initially be conducted by myself and 2 or 3 undergraduates. When CSU San Marcos begins its Master's degree program in 1997, I hope to have at least 2 graduate students (Masters) working in the lab. I also teach courses in Comparative Physiology, Human Physiology, Neurobiology, and Endocrinology. The equipment purchased with this grant will be used for demonstrations in conjunction with these courses. California State University at San Marcos has committed itself to offering a program in the Biological Sciences that stresses the use of modern equipment and the latest techniques. This commitment is shown in part by the generous set-up funds for a University of this size. However, the costs of acquiring the equipment necessary of neurophysiological research are beyond the capability of this institution. I therefore hope the NSF will support the university in its endeavor.
