Chemogenetic and optogenetic control of neuronal differentiation

目前的看法是，为了正常发育，神经元需要有电活动。事实上，缺乏活动会损害神经化学发育，例如，GAD或小白蛋白等功能蛋白的表达仍然很低或延迟。缺乏活动也会损害结构分化，因为轴突和突触的发育需要在发育的关键时期进行活动。在这里，我们建议激活和沉默单个神经元，并使用两种工具：光遗传工具通道视紫红质和具有抑制作用的代谢性化学遗传学(HM4Di)“由设计者药物激活的设计者受体”DREADD，通过在大鼠枕叶皮质切片培养中激活的神经元组来激活网络。氯氮平N-氧化物通过G蛋白和GIRK通道刺激dREADD hM4Di导致超极化。LED蓝光刺激通道-视紫红质以我们可以精确调节的频率和脉冲持续时间触发膜去极化。利用LED和CNO介导的刺激，我们将在几天内在不同的出生后时间窗重复触发去极化或超极化。我们预测，这将影响转基因神经元本身的发育，并在去极化刺激下导致整个网络的激活，进而可能进一步加速分化，这将通过形态计量学和蛋白质生物化学进行评估。到目前为止，DREADD和通道视紫红质都没有被用来指导发育过程。我们专注于篮状细胞，一种常见的皮质抑制性神经元类型，以及锥体细胞。我们将确定轴突分支、轴突大小和轴突密度、轴突起始节段的长度和位置、单个神经元的树突复杂性以及网络水平上中间神经元和突触分子标志物的表达是否可以受到重复去极化或超极化的调节。我们将确定这些操作是否在特定的发育时间窗口起作用，例如，神经元从超极化的化学握力释放后是否能从发育延迟中恢复。通道视紫红质方法提供了以选定的频率和脉冲持续时间以非侵入性方式刺激单个神经元的独特可能性。在这里，我们将确定结构和神经化学的发展，特别是中间神经元类型的发展是否可以通过重复去极化来调节，以测试去极化的频率或持续时间起到更重要的作用。

Current belief is that for proper development neurons need to be electrically active. Indeed, activity deprivation impairs neurochemical development, for instance, the expression of functional proteins like GAD or parvalbumin remains low or is delayed. Activity deprivation also impairs structural differentiation because neurite and synapse development requires activity during critical periods of development. We propose here to activate and to silence individual neurons, and to activate the network via sets of activated neurons in slice cultures of rat occipital cortex using two tools: the optogenetic tool channelrhodopsin and metabotropic chemogenetics with inhibitory (hM4Di) “designer receptors activated by designer drug”, DREADD. DREADD hM4Di stimulation with the specific ligand clozapine N-oxide results in hyperpolarization via a G-protein and GIRK channels. LED blue light stimulation of channelrhodopsin triggers membrane depolarizations at frequencies and pulse durations which we can exactly regulate. Using LED- and CNO-mediated stimulation we will trigger repetitively over a couple of days in different postnatal time windows depolarization or hyperpolarization. We predict that this will affect the development of the transfected neuron itself and – with depolarizing stimuli – result in an activation of the entire network which in turn could further accelerate differentiation, and which will be assessed by morphometry and protein biochemistry. Neither DREADDs nor channelrhodopsin have so far been explored to any big extend for steering developmental processes. We focus on basket cells, a frequent inhibitory neuron type in cortex, and pyramidal cells. We will determine if axonal branching, bouton size and bouton density, axon initial segment length and position, and dendritic complexity of individual neurons as well as expression of markers of interneurons and synaptic molecules at the network level can be regulated by repetitive depolarization or hyperpolarization. We will determine if these manipulations work in specific developmental time windows, and for instance, if neurons can recover from a developmental delay after being released from the hyperpolarizing chemical grip. The channelrhodopsin approach offers the unique possibility to stimulate individual neurons non-invasively with selected frequencies and pulse durations. Here, we will determine if structural and neurochemical development in particular of interneuron types can be regulated by repetitive depolarization, to test if frequency or duration of the depolarisation plays the more important role.