Acquisition of Electrophysiological Equipment to Study the Modulatory Influence of Circulating Hormones on Well-DefinedNeural Networks

这笔赠款将用于购买进行神经生理学实验的设备。该设备将由隔振台、微操作器、示波器、适当的放大器、磁带和图表记录器以及刺激器组成。使用该设备进行的研究将确定循环激素对明确定义的神经网络的精确影响。所使用的制剂将是刺龙虾的口胃神经系统(STNS)。STNS控制与胃相关的所有运动。在龙虾中，咀嚼发生在胃的胃磨区。口胃神经节(STG)尤其产生与胃运动相关的两种不同的运动模式。龙虾的蜕皮由两种循环激素控制--一组类固醇激素统称为蜕皮激素，另一种是多肽激素，称为蜕皮抑制激素(MIH)。在蜕皮周期中，MIH的循环水平下降，这触发了蜕皮类固醇的上升(图1)。一旦蜕皮激素水平达到峰值并开始下降，就会发生蜕皮。龙虾在蜕皮前停止进食，并在蜕皮期间脱落胃壁。因此，我的工作假设是，MIH在维持sTNS的日常功能方面发挥着至关重要的作用。我进一步预计，MIH水平的下降或蜕皮类固醇水平的增加，最初将抑制STG的运动模式。随后触发蜕皮的蜕皮类固醇水平的下降将反过来引发一种与胃壁脱落相关的新的运动模式。甲壳类动物的蜕皮可以通过去除幼年动物的眼柄来诱导。眼柄含有释放MIH的神经分泌细胞。切除眼柄会导致蜕皮类固醇的增加。因为动物本身正在制造激素，所以适当的蜕皮类固醇会以适当的比例产生。我将使用解剖学和生理学技术来检查循环激素对sTNS的影响。我的具体目标将是：目标1：确定带刺龙虾蜕皮周期中循环蜕皮类固醇的准确浓度。我已经开始与伯明翰阿拉巴马大学的D.Watson博士合作，他将进行放射免疫分析，以确定诱导蜕皮过程中蜕皮类固醇的循环水平。在蜕皮周期的不同阶段，将从动物身上提取血清样本，以确定蜕皮期间激素变化的时间进程。目的2：检测调节性神经递质表达的变化。北极癌早期实验的一个单一结果表明，在自然蜕皮过程中，某些调节递质的表达被下调。我将使用解剖学和免疫细胞化学技术来测量在去除眼柄和对照动物的sTNS中已识别的递质物质的表达。目的3：检测蜕皮前和蜕皮过程中sTNS运动模式和单个网络神经元的变化。为了做到这一点，我将结合细胞内和细胞外的记录来监测有节奏的运动模式。前期工作将包括对蜕皮诱导和对照动物的STG运动模式的细胞外和细胞内监测。在后面的实验中，我将使用电压钳技术来监测单个神经元中离子电导的变化。目的4：描述调制输入对运动模式产生网络的影响的变化。我之前进行的研究表明，调节性输入对STG的影响在自发蜕皮之前就会改变。我将使用已识别和表征的神经调节剂的沐浴应用以及对个人输入的刺激来比较蜕皮过程中和蜕皮之间的效果。这项工作最初将由我和2到3名本科生进行。当加州大学圣马科斯分校1997年开始攻读硕士学位时，我希望至少有两名研究生(硕士)在实验室工作。我还教授比较生理学、人类生理学、神经生物学和内分泌学的课程。用这笔赠款购买的设备将用于与这些课程一起进行演示。加州州立大学圣马科斯分校致力于开设生物科学课程，强调现代设备和最新技术的使用。这一承诺在一定程度上体现在为这样一所规模的大学提供慷慨的组建资金上。然而，购买神经生理学研究所需设备的成本超出了该机构的能力范围。因此，我希望NSF能支持这所大学的努力。

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.