| Title: |
Characterization of synaptic transmission in autaptic cultured neurons derived from human induced pluripotent stem cells |
| Authors: |
Fenske, Pascal |
| Contributors: |
male; Rosenmund, Christian; Sigrist, Stephan |
| Publication Year: |
2021 |
| Collection: |
FU Berlin: Refubium |
| Subject Terms: |
neurotransmitter release; synaptic transmission; human induced pluripotent stem cells; autaptic cultured neurons; human induced neurons; ddc:570 |
| Description: |
Human iNs derived from iPSCs hold great potential for modeling human neurological disorders and understanding their underlying causes. Until now, functional analysis of human iNs has been performed in conventional mass culture systems. Though useful for studying iN phenotypes, conventional cell culture lacks the ability to quantitatively characterize developmental and synaptic phenotypes at the individual cell level. That can be achieved with a more specialized cell culture system, such as the single neuron autaptic culture. Here, we developed a two-step protocol to generate autaptic cultures of iPSC-derived iNs using two independent iPSC lines and a neuronal induction protocol based on NGN2 expression. In the first experiments we show that our method efficiently generates mature, autaptic iNs with normal synapse densities. In these human iNs we were able to measure robust spontaneous and AP driven glutamatergic synaptic transmission. Furthermore, the sensitivity of synaptic responses to modulation by agonists of metabotropic receptors as well as potentiation by acute phorbol ester application. In the second part of this work we performed an application-based validation of the autaptic human iNs from human iPSCs. This allowed us to demonstrate the reliability of our cell culture system of human iNs as a replacement for animal models. As a proof of principle, we first used an acute shRNA based knocked down of UNC 13-A. Therefore, we added the knock down construct during the second phase of the newly developed protocol, which was only 14 days prior the experimental analysis in the autaptic cultures. We showed that the loss of UNC13A leads to a disruption of NT release. As a second approach we generated a human stem cell line deficient for Synaptotagmin 1, combined with the lentiviral reintroduction of Syt1. Using this approach, we tested the sensitivity of our protocol for the generation of human neural microcultures through the investigation of synaptic transmission in comparison to the known effect in mouse ... |
| Document Type: |
doctoral or postdoctoral thesis |
| File Description: |
99 Seiten; application/pdf |
| Language: |
English |
| DOI: |
10.17169/refubium-30618 |
| Availability: |
https://refubium.fu-berlin.de/handle/fub188/30879; https://doi.org/10.17169/refubium-30618; https://nbn-resolving.org/urn:nbn:de:kobv:188-refubium-30879-8 |
| Rights: |
http://www.fu-berlin.de/sites/refubium/rechtliches/Nutzungsbedingungen |
| Accession Number: |
edsbas.60FACEF |
| Database: |
BASE |