An optogenetic investigation to reveal the role and function of septohippocampal glutamatergic neurons in oscillations and memory

θ波振荡是一种主要的同步神经信号（3-12Hz），发生在哺乳动物大脑的海马体中，被认为在突触可塑性和情景记忆中起重要作用。虽然中隔被认为是通过从中隔-海马通路输入的谷氨酸能、胆碱能和gaba能产生θ活动所必需的，但这些神经元如何参与θ产生的机制尚不清楚。*该提议的中心假设是MS-DBB中的谷氨酸神经元在控制海马θ波振幅和提供整个海马的θ波振荡器同步方面至关重要。由于它们在调节θ节律中的作用，内侧间隔细胞将在学习和记忆中发挥关键作用。目的1是在解剖学和功能上确定视蛋白在中隔和海马表达的有效性。在这里，我们将确定增加谷氨酸神经元的放电速率是否足以增强海马theta。新兴的光遗传学技术将与我们的完整的隔海马体体外制备相结合，以操纵和研究神经元放电。光遗传学涉及引入两种光敏蛋白，ChETA用于选择性地使神经元去极化，Archt用于超极化。VGLUT2-CRE转基因小鼠将被注射ChETAor Archt cre重组酶AAV，特异性靶向MS-DBB的谷氨酸神经元（在出生后的早期），实验将使用间隔海马制备和间隔切片进行。在确定光参数以获得切片中内侧间隔谷氨酸神经元的最佳光激活后，我们将利用这些参数来确定海马中谷氨酸间隔神经元的作用。在海马CA1、CA3和海马体下进行全细胞和现场记录。局部场电位（LFPs）将使用傅里叶分析进行分析，频率和功率的变化将被量化。在前人的研究基础上，我们预测具有特定光遗传激活的谷氨酸神经元放电速率的增加将导致theta节律功率的增加。第二个目的是评估内侧间隔谷氨酸神经元在体外控制海马theta节律发生器中的作用。在以前的实验中已经观察到海马体中独立产生的θ波发生器。预计谷氨酸神经元提供了这些独立振荡的叠加和同步。在谷氨酸神经元的放电频率范围内记录CA1、CA3和下托区域的现场记录，并使用相干性和统计分析进行比较。这将使我们能够量化间隔谷氨酸能输入在多大程度上可以调节不同海马子区之间的θ同步以及实现这种调节所需的发射频率。最后，目的3是确定中隔谷氨酸神经元在体内调节海马依赖性记忆编码和回忆中的作用。*我们将利用这些结果在使用情境恐惧条件反射学习任务的学习任务中光遗传学调节内侧间隔谷氨酸神经元。我们假设激活内侧隔谷氨酸神经元会增强记忆编码，而沉默它们会损害记忆。*这些实验将首次解释内侧间隔谷氨酸神经元如何参与海马theta活动，以及对学习和记忆的重要性。

Theta oscillation is a dominant, synchronous neural signal (3-12Hz) that occurs in the hippocampus of the mammalian brain and is thought to play an important role in synaptic plasticity and episodic memory. Although the medial septum is thought to be necessary in generating theta activity through glutamatergic, cholinergic and GABAergic inputs from the septo-hippocampal pathway, the mechanisms underlying how these neurons are involved in theta generation is unknown.*The central hypothesis of this proposal is that glutamate neurons in the MS-DBB are critical in controlling the amplitude of hippocampal theta and for providing synchronization of theta oscillators throughout the hippocampus. Becase of their role in modulating theta rhythm, medial septal cells will play a critical role in learning and memory. Aim 1 is to determine anatomically and functionally the effectiveness of opsin expression in the medial septum and hippocampus. Here we will determine whether increasing the firing rate of glutamate neurons is sufficient to enhance hippocampal theta. The emerging technique of optogenetics will be combined with our complete septo-hippocampal preparation in vitro in order to manipulate and study neuronal firing. Optogenetics involves the introduction of two light-sensitive proteins, ChETA to selectively depolarize neurons, and Archt for hyperpolarization. VGLUT2-CRE transgenic mice will be injected with either a ChETAor Archt Cre-recombinase AAV, targeted specifically to glutamate neurons of the MS-DBB (at early postnatal days), and experiments will be conducted using the septo-hippocampal preparation as well as in septal slices. After determining the light parameters to obtain optimal light activation of medial septal glutamate neurons in slices, we will use these parameters to determine the role of glutamate septal neurons in hippocampus. Whole-cell and field recordings will be done in areas CA1, CA3 and subiculum of the hippocampus. The local field potentials (LFPs) will be analysed using Fourier analysis and changes in theta frequency and power will be quantified. Based on previous work it is predicted that increasing firing rate of glutamate neurons with specific optogenetic activation will lead to an increase in theta rhythm power. The second aim is to assess the role of medial septal glutamate neurons in controlling theta rhythm generators in hippocampus in vitro. Independently arising theta generators in the hippocampus have been observed in previous experiments. It is expected that glutamate neurons provide an overlying entrainment and synchronization of these independent oscillators. Field recordings in areas CA1, CA3 and subiculum will be recorded under a range of firing frequencies of glutamate neurons and compared using coherence and statistical analyses. This will allow us to quantify the degree to which septal glutamatergic input can modulate theta synchronization between different hippocampal subfields and the necessary firing frequency in order to achieve such modulation. Finally, aim 3 is to determine the role of septal glutamate neurons in modulating hippocampal-dependent memory encoding and recall in vivo.*we will use these results to optogenetically modulate medial septum glutamate neurons during a learning task using the contextual fear conditioning learning task. We hypothesize that activating medial septum glutamate neurons will enhance encoding of memory whereas silencing them will impair memory.* These experiments will provide for the first time an explanation of how medial septum glutamate neurons contribute to hippocampal theta activity and are important for learning and memory.