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

Theta振荡是一种主要的、同步的神经信号(3-12赫兹)，发生在哺乳动物大脑的海马区，被认为在突触可塑性和情景记忆中发挥着重要作用。尽管内侧隔区被认为是通过谷氨酸能、胆碱能和GABA能从隔膜-海马通路输入产生theta活动所必需的，但这些神经元如何参与theta产生的潜在机制尚不清楚。*这一提议的中心假设是，MS-DBB中的谷氨酸神经元在控制海马theta的幅度和提供整个海马theta振荡器的同步方面至关重要。由于内侧隔细胞在调节theta节律中的作用，它们将在学习和记忆中发挥关键作用。目的1是从解剖学和功能上确定视蛋白在内侧隔区和海马区表达的有效性。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。这项新兴的光遗传学技术将与我们完整的隔区-海马体体外准备相结合，以操纵和研究神经元放电。光遗传学包括引入两种光敏蛋白，Cheta用于选择性去极化神经元，ARCHT用于超极化。VGLUT2-CRE转基因小鼠将被注射ChETA或ARCHT CRE重组酶AAV，目标是MS-DBB的谷氨酸神经元(在出生后早期)，实验将使用隔区-海马区准备以及隔区切片进行。在确定了获得脑片内侧隔谷氨酸神经元最佳光激活的光参数后，我们将使用这些参数来确定谷氨酸隔神经元在海马区的作用。全细胞和现场记录将在海马区CA1、CA3和下丘脑进行。局域场势(LFP)将使用傅立叶分析进行分析，并且将量化theta频率和功率的变化。在前人工作的基础上，有人预测，具有特定光基因激活的谷氨酸神经元的放电频率增加将导致theta节律功率的增加。第二个目的是评估内侧隔区谷氨酸神经元在体外控制海马theta节律产生器中的作用。在以前的实验中已经观察到在海马体中独立地产生theta生成器。预计谷氨酸神经元为这些独立振荡器提供覆盖的夹带和同步化。在谷氨酸神经元的放电频率范围内，记录CA1区、CA3区和下丘脑区的现场记录，并使用相干和统计分析进行比较。这将使我们能够量化隔区谷氨酸能输入对不同海马亚区之间的theta同步的调制程度，以及实现这种调制所需的放电频率。最后，目标3是确定隔区谷氨酸神经元在体内调节海马依赖记忆编码和回忆中的作用。*我们将利用这些结果在学习任务中利用情境恐惧条件反射学习任务对隔区内侧谷氨酸神经元进行光遗传调制。我们假设，激活内侧隔区谷氨酸神经元将增强记忆的编码，而沉默它们将削弱记忆。*这些实验将首次解释内侧隔区谷氨酸神经元如何促进海马theta活动，并对学习和记忆至关重要。

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.