Chemogenetic and optogenetic control of neuronal differentiation

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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.