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

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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.