Collaborative Research: Glial regulation of extracellular potassium as an underlying developmental mechanism for the male-female difference in pacemaker neuron firing frequency

雄性和雌性之间的身体形态和行为差异在许多物种中都得到了很好的证实，但人们对它们在发育过程中是如何组织的知之甚少。棕色幽灵刀鱼的电通讯为阐明其中一种差异的细胞机制提供了一个独特的机会。在这种弱电鱼中，雄鱼产生的电子器官放电频率高于雌鱼。放电频率的差异是由起搏核控制的，起搏核是脑干中的一个神经回路。使用跨学科的策略，将检验一个新的机制假设：星形胶质细胞(一种胶质细胞类型)对细胞外钾离子的调节(缓冲)在男性和女性中是不同的，这是他们电子器官放电差异的原因。除了为男性和女性之间的一种特殊行为所依据的分子和细胞机制提供新的见解外，这项研究还将促进对星形胶质细胞在神经系统钾缓冲中的作用以及这些离子对神经网络的调节的了解。后两个主题在涉及模式产生神经回路的病理条件的病因学中发挥着重要作用(例如，高频癫痫发作被认为是由于钾缓冲受损，从而增强了此类癫痫发作的易感性)。由于这项研究的跨学科性质，还为不同学科的学生培训以及生物学与其他STEM学科之间的课程开发提供了多种机会。本科生将通过东北大学和弗吉尼亚大学提供的指导学习课程参与实施这一研究项目；东北大学的学生还将获得为期6个月的实习机会，以参与所进行的项目和弗吉尼亚大学的部分项目。国际和平研究所和共同国际和平研究所还将把这项研究的一部分用于他们在各自机构教授的以实验室为基础的高级本科课程，以及参与当地高中的外联活动和面向普通公众的一系列STEM活动。弱电鱼类Apteronotus eptorhynchus的电器官放电是一个健壮的、具有良好特征的行为系统。这些放电的频率在男性和女性之间确实是不同的，并受起搏核的神经活动控制，起搏核是延髓中的一种内源性振荡器。参与这些振荡产生的关键神经元是起搏器和中继细胞，它们被星形细胞合胞体所包围。在这些星形胶质细胞的形态、胶质细胞特异性蛋白的表达以及合体内单个细胞的缝隙连接耦合方面存在显著的男性和女性差异。这项研究将验证这样一种假设，即起搏器核中细胞外钾离子的星形细胞缓冲能力调节了电子器官放电频率的男女差异的发展，从而导致细胞外钾浓度的变化，最终改变了由起搏器和中继细胞组成的神经网络的振荡频率。为了实现这一目标，将采用多学科和跨学科的方法，包括星形细胞合胞体与起搏器和中继细胞的联系的形态和分子分析；神经胶质功能的药理学操作和行为效应的检查；星形胶质细胞控制起搏器核放电频率的离子机制的电生理表征；以及对(I)星形细胞合胞体对钾离子的摄取和再分配，(Ii)这一作用对起搏器核团神经振荡频率的影响的计算模拟。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Differences in body form and behavior between males and females are well established in numerous species, but little is known about how they are organized during development. Electric communication by the brown ghost knifefish provides a unique opportunity for elucidating the cellular mechanisms mediating one such difference. In this weakly electric fish, males produce electric organ discharges that contain higher frequencies than those of females. The difference in discharge frequencies is controlled by the pacemaker nucleus, a neural circuit in the brainstem. Using an interdisciplinary strategy, a novel mechanistic hypothesis will be tested: astrocytes (a glial cell type) regulate (buffer) extracellular potassium ions differently in males and females, and this is responsible for the difference in their electric organ discharges. Besides providing new insights into the molecular and cellular mechanisms that underlie one specific behavior that is dimorphic between males and females, this research will also advance knowledge about the role of astrocytes in potassium buffering in the nervous system, and about the regulation of neural networks by these ions. The latter two topics play an important role in the etiology of pathological conditions involving pattern-generating neural circuits (for example, high-frequency epileptic seizures are thought to result from an impairment in potassium buffering that enhances the susceptibility for such seizures). Because of the interdisciplinary nature of this research, multiple opportunities are also provided for training students in diverse disciplines, and for curriculum development at the interface between biology and other STEM disciplines. Undergraduates will be involved in carrying out this research project via directed study courses being offered at both Northeastern University and the University of Virginia; 6-month internships will also be offered for students from Northeastern University to participate in the parts of the project conducted and at the University of Virginia. The PI and co-PI will also use parts of this research in laboratory-based upper-level undergraduate courses that they teach at their respective institutions, as well as engaging in outreach activities at local high schools and a series of STEM events for the general public. The electric organ discharge of the weakly electric fish Apteronotus leptorhynchus is a robust and well-characterized behavioral system. The frequency of these discharges is reliably different in males and females, and controlled by the neural activity of the pacemaker nucleus, an endogenous oscillator in the medulla oblongata. The neurons critically involved in the generation of these oscillations are the pacemaker and relay cells, which are enveloped by astrocytic syncytia. Significant male-female differences exist in the morphology of these astrocytes, their expression of glia-specific proteins, and the gap-junction coupling of individuals cells within the syncytium. The study will test the hypothesis that the development of the male-female difference in the frequency of the electric organ discharge is regulated through modulation by the astrocytic buffering capacity of extracellular potassium ions in the pacemaker nucleus, thereby resulting in changes in the extracellular potassium concentration and ultimately in the oscillation frequency of the neural network composed of pacemaker and relay cells. To achieve this objective, a multi- and interdisciplinary approach will be employed, including morphological and molecular analysis of the association of the astrocytic syncytium with the pacemaker and relay cells; pharmacological manipulation of glial function and examination of its behavioral effect; electrophysiological characterization of the ionic mechanisms of astrocytes that control the firing frequency of the pacemaker nucleus; and computational modeling of (i) the uptake and redistribution of potassium ions by the astrocytic syncytium, (ii) the effect of this action on the frequency of the neural oscillations of the pacemaker nucleus.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.