Role of astrocytes in neuromodulator-mediated shaping of cortical networks

基本原理：神经调质通过影响皮层抑制来影响神经回路内的信噪比和频率传输。由于许多神经调质以突触外体积的方式释放，其中降解/摄取和复杂CNS的限制限制了扩散距离，我们提出了这样一个问题：星形胶质细胞是否迅速将神经调质作用扩展到每个突触？星形胶质细胞理想地位于延长神经调质作用的位置，因为每个星形胶质细胞可以1）包封超过100，000个突触，2）释放已知影响抑制的ATP/腺苷，3）调节可以影响兴奋性的细胞外钾，以及4）表达所有神经调质的受体。了解星形胶质细胞在神经调节剂介导的行为状态调节中的作用将突出其在精神病和神经退行性疾病中的重要性。在这里，我们探讨了星形胶质细胞通过改变钙动力学、嘌呤释放和钾稳态来延长神经调节作用的假设。 目的1：用全细胞膜片钳技术研究神经调质对皮层第二层神经元自发性和诱发性突触后电流的影响。使用GAPDH抑制剂和各种嘌呤能激动剂/拮抗剂将帮助我们更好地了解星形胶质细胞糖酵解和嘌呤能信号传导在神经调节剂介导的皮质网络变化中的作用。用钙螯合剂直接进行星形胶质细胞贴片透析将进一步能够直接评估任何星形胶质细胞的作用，而不影响周围的细胞或突触。 目的2将评估嘌呤释放和细胞外钾调节在神经调节剂介导的抑制效应中的作用。快速扫描循环伏安法将评估神经调节剂对ATP/腺苷释放的影响。钾离子选择性电极将用于测量神经调节剂介导的对基线和活性介导的细胞外钾变化的影响。 目标3将使用双光子成像评估神经调节剂对皮质切片星形胶质细胞钙动力学的影响，使用由腺相关病毒递送的遗传编码的钙指示剂。我们将评估在控制和神经调节剂条件下诱发钙事件的阈值（20-50 Hz的刺激次数）和动力学（速度/距离/振荡）。 意义：星形胶质细胞功能障碍、应激和神经调质丢失与神经退行性疾病和精神疾病的发展相关。了解星形胶质细胞在生理条件下协调网络反应的作用是了解与压力，神经退行性变和精神疾病相关的病理学的必要的第一步。此外，这些研究支持精神病学的根本转变，通常侧重于神经回路到神经元-胶质细胞相互作用，可能扩大药物治疗的基础。

Rationale: Neuromodulators affect signal-to-noise and frequency transmission within a neural circuit by effects on cortical inhibition. As many neuromodulators are released in an extrasynaptic volume manner where degradation/uptake and the confines of the complex CNS limit diffusion distance, we ask the question Do astrocytes rapidly extend neuromodulator actions to every synapse? Astrocytes are ideally situated to extend neuromodulator effects given that each astrocyte can 1) ensheath over 100,000 synapses, 2) release ATP/adenosine known to affect inhibition, 3) regulate extracellular potassium that can affect excitability and 4) express receptors for all neuromodulators. Understanding the role astrocytes play in neuromodulator-mediated regulation of behavioral state will highlight their importance in psychiatric and neurodegenerative conditions. Here, we explore the hypothesis that astrocytes extend neuromodulatory influence via alterations in calcium dynamics, release of purines, and potassium homeostasis. Aim 1 will examine neuromodulator effects on spontaneous and evoked postsynaptic currents in isolation by whole-cell patch-clamp of layer II neurons in cortical slices. Use of a GAPDH inhibitor and various purinergic agonists/antagonists will help us better understand the role astrocyte glycolysis and purinergic signaling play in neuromodulator-mediated changes to cortical networks. Direct astrocyte patch dialysis with a calcium chelator will further enable direct assessment of any astrocytic role without affecting surrounding cells or synapses. Aim 2 will assess the role of purine release and regulation of extracellular potassium in neuromodulator-mediated effects on inhibition. Fast scan cyclic voltammetry will assess neuromodulator effects on ATP/adenosine release. Potassium ion-selective electrodes will be used to measure neuromodulator-mediated effects on both baseline and activity-mediated changes in extracellular potassium. Aim 3 will use two-photon imaging to assess neuromodulator influence on astrocyte calcium dynamics in cortical slices using genetically-encoded calcium indicators delivered by an adeno-associated virus. We will assess both the threshold (number of stimulations at 20-50 Hz) and dynamics (speed/distance/oscillations) of evoked calcium events under control and neuromodulator conditions. Significance: Astrocyte dysfunction, stress and neuromodulator loss correlate with development of neurodegenerative and psychiatric disorders. Understanding the role of astrocytes in coordination of network responses under physiological conditions is a necessary first step to understanding pathology associated with stress, neurodegeneration, and psychiatric disorders. Furthermore, these studies support a fundamental shift in psychiatry that typically focuses on neural circuits to that of neuron-glia interactions, potentially expanding the basis for pharmacological treatment.