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

θ振荡是一种发生在哺乳动物大脑海马区的主导同步神经信号（3- 12 Hz），被认为在突触可塑性和情景记忆中起重要作用。虽然内侧隔被认为是通过隔-海马通路的多巴胺能、胆碱能和γ-氨基丁酸能输入产生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theta的振幅和在整个海马中提供theta振荡器的同步方面是至关重要的。由于它们在调节θ节律中的作用，内侧隔细胞将在学习和记忆中起关键作用。目的1是从解剖学和功能上确定视蛋白在内侧隔和海马中表达的有效性。在这里，我们将确定增加谷氨酸神经元的放电率是否足以增强海马theta。新兴的光遗传学技术将与我们完整的隔-海马体外制备相结合，以操纵和研究神经元放电。光遗传学涉及两种光敏蛋白的引入，ChETA选择性地使神经元去极化，而Archt用于超极化。VGLUT 2-CRE转基因小鼠将注射ChETA或Archt Cre重组酶AAV，特异性靶向MS-DBB的谷氨酸神经元（在出生后早期），并将使用隔-海马制备物以及隔切片进行实验。在确定光参数以获得切片中内侧隔谷氨酸神经元的最佳光激活之后，我们将使用这些参数来确定谷氨酸隔神经元在海马中的作用。将在海马CA 1、CA 3和下托区域进行全细胞和视野记录。将使用傅立叶分析分析局部场电位（LFP），并量化θ频率和功率的变化。基于先前的工作，预测具有特定光遗传学激活的谷氨酸神经元的放电速率的增加将导致θ节律功率的增加。第二个目的是评估内侧隔谷氨酸神经元在体外控制海马θ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出现的θ发生器。预期谷氨酸神经元提供这些独立振荡器的叠加夹带和同步。将在谷氨酸神经元的一系列放电频率下记录区域CA 1、CA 3和下托中的场记录，并使用相干性和统计分析进行比较。这将使我们能够量化的程度，隔海马神经元输入可以调制不同的海马子字段之间的θ同步和必要的发射频率，以实现这种调制。最后，目标3是确定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。我们将使用这些结果在使用情境恐惧条件化学习任务的学习任务期间光遗传学地调节中间隔谷氨酸神经元。我们假设，激活内侧隔谷氨酸神经元会增强记忆的编码，而沉默它们会损害记忆。这些实验将首次解释内侧隔谷氨酸神经元如何促进海马theta活动，并对学习和记忆很重要。

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.