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Chemogenetic and optogenetic control of neuronal differentiation

Subject Area Developmental Neurobiology
Term from 2018 to 2022
Project identifier Deutsche Forschungsgemeinschaft (DFG) - Project number 406298856
 
Final Report Year 2023

Final Report Abstract

We could not realize some of the smaller goals proposed in the grant. The plan to analyze the effects of hM3Ds (chimeric m3/β1 adrenergic receptor stimulating a Gαs pathway) was canceled in order to focus in depth on the muscarinic receptor variants 4i and 3q. Also, the plan to characterize the co-action of GIRK2 had to be cancelled for reasons of time, personnel, and work restrictions during the reporting period. On the brighter side, the plan to run protein blots with optogenetic stimulation has in fact been done. Steffen Gonda quantified the expression of selected proteins (GAD65, GAD 67, Synaptotagmin-2, Synapsin-1, GluN2B, GluN2B Y1472 phosphorylation, GluA1, GluA1 S831 phosphorylation) in lysates (min. 11, max. 35 cultures, no technical repeats) of cultures optogenetically stimulated @0.5 Hz. Only one turned out to be altered: GluA1 total protein was significantly downregulated upon 70 ms and even stronger upon 140 ms flash duration. Our earlier work has identified GluA1 as major driver of apical dendritic growth of pyramidal cells. Thus, the protein blot result of less GluA1 is in line with the finding of stunted apical growth of pyramidal cell subsets after repetitive optogenetic stimulation. The plan to look at the dynamics of the axon initial segment in cortical neurons with higher or lower levels of activity had to be modified because the dynamics is under investigation in the labs of collegues. Instead, we looked at the question of whether or not silenced or more active pyramidal neurons have more or less AcD and if these AcD grow to a degree of complexity that differs from that of regular dendrites. For this we began to exploit the slice culture material produced over the past decades with two bachelor projects. Yet, it is still too preliminary to claim a story. Yet, towards that direction, we studied the AcD phenotype in 7 mammalian species (mouse, rat, cat, ferret, pig (wild boar), macaque, human), with various methods (Lucifer Yellow injections, biocytin tracing, Golgi, immunohistochemistry and confocal analysis, genetic labeling). By analyzing ~34.000 neurons of primary sensory and higher order cortical areas mostly in monkey we discovered remarkable species differences in the proportion of neurons displaying the AcD configuration. Human pyramidal cells of supragranular layers have the lowest number of AcD. Together, we managed to obtain substantial novel information on how activity shapes cortical neurons, and that “more activity” does not always lead to in “higher complexity”. Personally, I am extremely happy with the unexpected original finding that an axonal origin from a dendrite increases and/or stabilizes dendritic complexity of interneurons and triggers the development of a denser local terminal field of basket neurons. Future work will show if this may result in stronger axosomatic inhibition of surrounding pyramidal cells.

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