Clinically unscreened vasculo-glial-neuronal coupling is critical for physiological brain function

虽然已经投入了大量的努力来理解与活动引起的变化有关的机制 脑血流量(CBF)即功能性充血和神经血管偶联，目前了解较少 控制基础脑血流量和静息神经元活动的过程。考虑到慢性脑低灌注 对认知障碍的贡献我们小组有兴趣研究细胞机制 稳态血管张力的变化，从而影响静息的神经元功能。我们的中央 假设定义生理性血管-胶质-神经元偶联(VGNC)的本构机制是 在疾病中受损的。使用多学科方法，我们将实现以下三个目标：目标1：测试 疾病中星形胶质细胞钙信号异常损害VGNC的假说。星形胶质细胞构成 整合大脑的灌流状态，通过释放神经胶质递质信号，调节静息神经元 相应的活动。VGNC的损伤使大脑面临谷氨酸兴奋毒性、炎症和 氧化应激。利用GLAST-CreERT2；R26-LSL-GCaMP3小鼠(体内和体外)，我们将测量星形细胞 ↑或↓引起管腔实质小动脉血管反应性改变时的钙离子事件 对照组和脑低灌注(双侧颈总动脉狭窄)脑片的血流/压力 老鼠。将使用药理学方法来确定关键的信号机制(即，P2Y1和TRPV4通道) 调停VGNC。AIM2.检验血管反应性变化直接相关的假设 静息状态下皮质锥体神经元活动的改变。最佳的能量平衡要求 神经活动的强度与血液灌注量适当匹配。使用体内和体外方法，我们将 在对照组中确定压力/流量实质小动脉直径的变化如何影响静息神经元活动 血管紧张素II依赖性高血压和脑血管疾病模型的建立 低灌注率。体外：测量小动脉直径，神经元膜电位，放电率和 在血流动力学刺激(如↑或↓血流/压力)之前、期间和之后获得突触电流 唤起了对PA的加压。体内：静息神经元活动对全身诱发的血液变化的反应 将对压力进行评估。Aim3.验证这样的假设，即血管反应性的变化通过 星形胶质细胞钙依赖通路、GABA能中间神经元调节皮质神经元网络。 利用脑实质小动脉直径与神经电生理活动的同步变化 我们将确定压力/流量引起的实质小动脉血管反应性变化的影响 对皮质GABA能中间神经元功能和神经元网络有影响。具体来说，我们将标识 GABA能中间神经元亚型在VGNC中驱动神经网络反应，无论是中间神经元 反应需要星形胶质细胞的钙变化作为中间步骤，以及中间神经元的反应是否 在疾病条件下改变的。 1

While much effort has been devoted to the understanding of mechanisms linked to activity-evoked changes in cerebral blood flow (CBF) namely, functional hyperemia and neurovascular coupling, less is understood about the processes controlling basal CBF and resting neuronal activity. Considering that chronic brain hypoperfusion contributes to cognitive impairments our group is interested in studying the cellular mechanisms by which changes in steady-state vascular tone, and thus perfusion, affect resting neuronal function. Our central hypothesis is that constitutive mechanisms defining physiological vasculo-glial-neuronal coupling (VGNC) are impaired in disease. Using a multidisciplinary approach we will address the following three aims: Aim1: Test the hypothesis that aberrant astrocytes Ca2+ signaling in disease impairs VGNC. Astrocytes constitutively integrate perfusion status to the brain and, through the release of gliotransmitter signals, adjust resting neuronal activity accordingly. Impairments in VGNC places the brain at risk for glutamate excitotoxicity, inflammation and oxidative stress. Using GLAST-CreERT2; R26-lsl-GCaMP3 mice (in vivo and in vitro) we will measure astrocytic Ca2+ events in response to parenchymal arteriole vascular reactivity changes evoked by ↑or↓ in lumen flow/pressure in brain slices from control and cerebral hypoperfused (bilateral common carotid artery stenosis) mice. A pharmacological approach will be used to define key signal mechanisms (i.e. P2Y1 and TRPV4 channel) mediating VGNC. Aim2. Test the hypothesis that changes in vascular reactivity are directly associated with changes in resting cortical pyramidal neuron activity. Optimal energy balance requires that the degree of neuronal activity be properly matched with blood perfusion. Using in vivo and in vitro approaches, we will determine how pressure/flow parenchymal arteriole diameter changes impact resting neuronal activity in control mice and in models of vascular disease using Angiotensin II-dependent hypertension and cerebral hypoperfusion. In vitro: measurements of arteriolar diameter, neuronal membrane potential, firing rates and synaptic currents are obtained before, during and after a hemodynamic challenge (e.g. ↑or↓ flow/pressure) evoked to pressurized PA. In vivo: resting neuronal activity in response to systemic-evoked changes in blood pressure will be assessed. Aim3. Test the hypothesis that changes in vascular reactivity recruit, via an astrocyte Ca2+-dependent pathway, GABAergic interneurons to regulate cortical neuronal networks. Using simultaneous parenchymal arteriole diameter changes with electrophysiological neuronal activity recordings we will determine the effect pressure/flow-evoked parenchymal arteriole vascular reactivity changes has on cortical GABAergic interneuron function and neuronal networks. Specifically, we will identify the GABAergic interneuron subtype driving neuronal network responses during VGNC, whether interneuron responses require astrocyte Ca2+ changes as an intermediate step and whether interneuron responses are altered in disease conditions. 1