Chemogenetic and optogenetic control of neuronal differentiation

目前的观点是，为了正常发育，神经元需要具有电活性。事实上，活动剥夺会损害神经化学物质的发育，例如，GAD或小白蛋白等功能蛋白的表达保持低水平或延迟。活动剥夺还会损害结构分化，因为神经突和突触的发育在发育的关键时期需要活动。我们在此建议激活和沉默单个神经元，并通过大鼠枕皮质切片培养中的激活神经元集激活网络，使用两种工具：光遗传学工具通道视紫红质和代谢化学遗传学与抑制性（hM4Di）“由设计药物激活的设计受体”，DREADD。特定配体氯氮平n -氧化物刺激DREADD hM4Di可通过g蛋白和GIRK通道产生超极化。LED蓝光刺激通道视紫红质触发膜去极化的频率和脉冲持续时间，我们可以精确地调节。使用LED和cno介导的刺激，我们将在几天内在不同的产后时间窗触发重复的去极化或超极化。我们预测这将影响转染神经元本身的发育，并且-通过去极化刺激-导致整个网络的激活，从而进一步加速分化，这将通过形态测定学和蛋白质生物化学进行评估。到目前为止，无论是DREADDs还是通道视紫质都没有在指导发育过程方面进行任何深入的探索。我们将重点放在篮状细胞和锥体细胞上，篮状细胞是皮层中常见的抑制性神经元类型。我们将确定轴突分支、钮扣大小和钮扣密度、轴突初始段长度和位置、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记物的表达是否可以通过重复去极化或超极化来调节。我们将确定这些操作是否在特定的发育时间窗口中起作用，例如，如果神经元在从超极化化学控制中释放出来后能够从发育延迟中恢复过来。通道视紫红质方法提供了独特的可能性，可以通过选择频率和脉冲持续时间非侵入性地刺激单个神经元。在这里，我们将确定结构和神经化学发育，特别是中间神经元类型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是否起着更重要的作用。

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.