Abstract
Recently developed technology to differentiate induced pluripotent stem cells (iPSCs) into human induced neurons (iNs) provides an exciting opportunity to study the function of human neurons. However, functional characterisations of iNs have been hampered by the reliance on mass culturing protocols which do not allow assessment of synaptic release characteristics and neuronal morphology at the individual cell level with quantitative precision. Here, we have developed for the first time a protocol to generate autaptic cultures of iPSC-derived iNs. We show that our method efficiently generates mature, autaptic iNs with robust spontaneous and action potential-driven synaptic transmission. The synaptic responses are sensitive to modulation by metabotropic receptor agonists as well as potentiation by acute phorbol ester application. Finally, we demonstrate loss of evoked and spontaneous release by Unc13A knockdown. This culture system provides a versatile platform allowing for quantitative and integrative assessment of morphophysiological and molecular parameters underlying human synaptic transmission.
Highlights
Autaptic cultures, in which single neurons grow in isolation on astrocytic microislands and form synapses exclusively with themselves[11], provide an experimental system that allows the quantitative assessment of input and output properties of individual neurons, both in morphological and in functional experiments[12]
We differentiated induced pluripotent stem cells (iPSCs) into hiNs using the protocol described by Zhang and colleagues[5] and plated the hiNs onto mouse-derived astrocytic microislands on day 4 post induction (DPI)
We adapted the differentiation protocol by splitting it into two phases: in the first phase we induced hiNs from iPSCs by forced expression of Ngn[2] as described (Zhang et al, 2013) and allowed them to develop as mass culture
Summary
In which single neurons grow in isolation on astrocytic microislands and form synapses exclusively with themselves[11], provide an experimental system that allows the quantitative assessment of input and output properties of individual neurons, both in morphological and in functional experiments[12]. We used whole-cell patch-clamp recordings in order to examine the passive and active membrane properties of the autaptic hiNs after 14 to 21 days in autaptic culture (Fig. 1B).
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