Evolution of synaptic activity due to AP-independent release of neurotransmitters over time in iPSC-derived neurons plated on POL.

<p>A) Example of continuous recording from an iPSC-derived neuron at 55 days (holding potential at −70 mV). The inset shows an enlarged view of two spontaneous post-synaptic events. B) The number of cells showing synaptic events over the total number of cells recorded in the same day. Cells were recorded at −70 mV in 1 µM TTX in voltage clamp. C) Increase over time of the frequency of the mEPSCs, measured as number of spontaneous events/minute. D) Evolution of the mEPSC amplitude with a voltage clamp at −70 mV from t1 to t3.</p

Spontaneous calcium transients increase in iPSC-derived neurons during development.

<p>A) After imaging of spontaneous Ca²⁺- transients iPSC-derived neurons were fixed and stained for Tuj1 at day 32 and day 55, revealing more complex morphology at day 55 (scale bar = 40 µm). B) Representative Fluo-4 spontaneous Ca²⁺- transients from 3 iPSC-derived neurons at day 32 and at day 55 (ΔFluo = ΔFluorescence; a.u. = arbitrary unit). Neurons exhibit slow broad transients, consistent with early developing neurons at day 32. By day 55, calcium transients take on a more spike-like morphology reflecting what has been reported for maturing neurons. Group data demonstrates that iPSC-derived neurons exhibit a significant increase in calcium transients at day 55 as compared to day 32 (n = 113 neurons/10 coverslips for both groups, p<0.001 comparing day 32 with day 55). C) The average number of Ca²⁺ transients at day 55 was dramatically reduced following perfusion with 1 µM TTX with recovery following washout of the toxin (n = 20/3 coverslips, p<0.001 compared with average number of Ca²⁺ transients prior to TTX perfusion). A sample trace derived from a representative experiment is shown on the left).</p

iPSCs-derived neurons plated with glia express different electrophysiological properties than neurons plated on POL.

<p>A) Representative APs in response to step current injections of 20 pA in current clamp mode for a cell at day 55 co-cultured with mouse glia. APs were observed on 13 out of 15 cells, and in 4 out of the 13 cells with APs rebound APs at the end of hyperpolarizing current injections were also present. B) Examples of mEPSCs recorded in voltage clamp configuration. Cells were plated on mouse glia by mixing them before plating. They were held at −70 mV. TTX at a concentration of 1 µM was added to the bath solution to suppress spontaneous excitation and to allow isolation of synaptic events induced by spontaneous transmitter release.</p

Characterization of the APs in iPSC-derived neurons plated on POL.

<p>A) Representative APs in response to step current injections of 20 pA (upper panel) in current clamp mode. The cell shown was recorded at day 45. APs were observed in 4 out of 10 cells. Following initial recording, APs were blocked by 1 µM TTX. B) Multiple firing of APs in response to step current injections in 20 pA increments was observed in cells from day 45. The trace shown in this figure was obtained from a cell recorded at 55 days. The inset shows a high magnification view of the APs. C) At day 55, 13 out of 22 cells had APs, and 3 of them had rebound APs like the one shown on the inset of this panel. Multiple APs in response to depolarizing current injections and “rebound” APs at the end of hyperpolarizing current injections were also visible. After perfusion with 1 µM TTX, the APs, due to Na⁺ channel dependence, was blocked, but rebound APs persisted in 4 out of 17 cells with rebound APs.</p

Flow cytometry and immunofluorescence analyses of iPSC-derived neurons.

<p>A) Flow cytometry analysis of neuronal surface markers CD56 and CD24 at day 32 of differentiation. B) Representative phase contrast microscope image of cultured neurons at day 32 of differentiation. Scale bar: 100 µm. C) At day 45 of differentiation, cells express MAP2 (green) along soma and dendrites and the postsynaptic protein PSD95 (red), confirming the advanced stage of differentiation into neurons. Scale bar: 100 µm. D) Cells also express the forebrain-specific marker BF1 (green) and Tuj1 (red) at 45 days of culture. They are often co-localized (yellow). Scale bar: 50 µm.</p

Timeline of the experiments.

<p>The different steps of the experiments are represented, as well as the time-points for the electrophysiological, calcium imaging, immunofluorescence and FACS analyses. The 3 different timing groups for statistical purposes are the following: t1  =  from day 31 to day 38; t2  =  from day 41 to day 45; t3  =  from day 48 to day 55.</p

Evolution of basal membrane properties over time in iPSC-derived neurons plated on POL.

<p>A) The average resting membrane potential (RMP in mV) decreased over time. The number of cells examined for these experiments are shown inside the bar in these and the following figures. The vertical lines correspond to the SEM in this and the following figures. **: p<0.001; *: 0.0010.05 for these and the following figures. B) The membrane input resistance decreased over time (GΩ). C) The membrane time constant decreased over time (ms).</p

Evolution of active membrane properties over time in iPSC-derived neurons plated on POL.

<p>A) Representative traces of voltage clamp recordings showing fast inward currents followed by long-lasting outward currents in a cell at day 55, due to voltage steps in 10 mV increments (shown in the upper panel). The inset shows a high magnification view of the inward current. Inward Na⁺ currents were observed in 13 out of the 22 cells. Following initial recording, cells were perfused with 1 µM TTX to block Na⁺ currents, and subsequently with 10 mM TEA (tetraethylammonium) to block K⁺ currents. B) The percentage of cells showing APs in response to step current injections of 20 pA over the total number of cells recorded in the same day. C) The graph shows the increase over time of the average amplitude of the inward Na⁺ current measured at the peak (pA). D) The graph shows a decrease over time of the Full Width at Half Maximum (FWHM) (ms) of APs. E) The graph shows an increase of the AP amplitude (mV) over time. F) Evolution of the AP threshold (mV) over time. G) Evolution of the average peak K⁺ currents over time (pA).</p

Electrophysiological parameters in neurons plated on POL and mouse glia co-cultures at day 55.

<p>The table summarizes the differences between various electrophysiological properties in neurons plated on POL (n = 20) and mouse-glia co-cultures (n = 15) at day 55. Values are expressed as mean and SE (standard error).</p