iNGNs mature in long-term culture.

<p>(A) Cell culture protocol for deriving long-term cultures of iNGNs. Astrocytes (ACs) were plated onto coverslips between 1 and 3 days before iNGNs. (B) Representative DIC microscope images of iNGNs were taken over the course of long-term co-culture with astrocytes, from 1–112 days. Ages (in days) are labeled in the upper left corners of the images. The uppermost left image shows iNGNs that are undifferentiated (no Dox). (C) Intrinsic properties of iNGNs from 4 to 70 days were measured in whole-cell patch-clamp recordings, and averaged measurements are shown for cell capacitance, resting membrane potential, and input resistance. Error bars denote SEM and numbers next to the errors bars show the number of cells recorded at each age.</p

Active properties of iNGNs in long-term culture.

<p>(A) Representative current-clamp traces showing action potentials from 7d, 28d, and 56d iNGNs, in response to a 500 ms pulse of current injection from a starting membrane potential of -75 mV. (B) Firing properties of iNGNs from 4 to 70 days in response to current injections of different amplitudes. The numbers of APs were summed over a 500 ms stimulus starting from a membrane potential of -75 mV. Numbers at the top of the histogram bars show the number of cells recorded at each age. (C) Average numbers of APs from 7d, 28d, and 56d iNGNs, in response to current injections up to +60 pA. (D) Action potential properties of iNGNs from 7 to 70 days. Average AP peak amplitudes (top histogram), average AP width at half the maximal peak (half-width, middle histogram), and average AP threshold (bottom histogram) in iNGNs from 7 to 70 days age. Error bars denote SEM and numbers next to the errors bars show the number of cells recorded at each age.</p

Spontaneous postsynaptic currents (sPSCs) in iNGNs.

<p>(A) Representative current traces in a 63d iNGN, showing sPSCs before (left trace) or mEPSCs after (right trace) addition of 1 μM tetrodotoxin (TTX) and 100 μM picrotoxin (PTX) to the ACSF bath solution. Voltage-clamp recordings were done at -75 mV. (B) Fractions of neurons with sPSCs in iNGNs from 4 to 70 days. Numbers at the top of the histogram bars show the number of cells recorded at each age. (C) The average frequency of sPSCs in iNGNs from 4 to 70 days. Error bars denote SEM and numbers next to the errors bars show the number of cells recorded at each age. (D) Fractions of neurons with mEPSCs in iNGNs from 4 to 42 days. Numbers at the top of the histogram bars show the number of cells recorded at each age. (E) The average frequency of mEPSCs in iNGNs from 14 to 42 days. Error bars denote SEM and numbers next to the errors bars show the number of cells recorded at each age.</p

Averaged mEPSC parameters.

<p>Averaged mEPSC parameters.</p

AMPA and KA receptor-mediated mEPSCs in iNGNs.

<p>(A) Representative current traces in a 21d iNGN, showing mEPSCs in ACSF bath solution containing 1 μM tetrodotoxin (TTX) + 100 μM picrotoxin (PTX, left trace), 1 μM TTX + 100 μM PTX + 50 μM CNQX (middle trace), and a subsequent wash with 1 μM TTX + 100 μM PTX (right trace). Voltage-clamp recordings were done at -75 mV. (B) Summaries of the number of mEPSC events before or after addition of 50 μM CNQX to 21d, 28d, 35d, and 42d iNGNs. Gray open circles represent the total number of events recorded from one cell and black closed circles are averages of all cells recorded for each age, with error bars denoting SEM and n being the number of cells recorded at each age. The number of events was different before and after addition of CNQX (21d iNGNs: **P = 0.0098, n = 7; 28d iNGNs: *P = 0.0224, n = 6; 35d iNGNs: *P = 0.0403, n = 7; 42d iNGNs *P = 0.0383, n = 6; paired Students <i>t</i>-tests).</p

iNGNs expressing ChR2(L132C) can be activated with light.

<p>(A) Representative voltage-clamp recording of a 23d iNGN expressing ChR2(L132C)-mKateA (by AAV2 transduction), in response to a 500 ms pulse of 36 mW/mm² 473 nm blue light at a membrane potential of -75 mV (upper trace). Representative current-clamp recording of the same iNGN, in response to a 500 ms pulse of 3.6 mW/mm² 473 nm blue light. Membrane potential was held at -75 mV (lower trace). (B) ChR2(L132C) steady-state current densities, in response to saturating light intensities (20–40 mW/mm²), in rat hippocampal neurons (n = 9, 13–26 DIV HCNs) or iNGNs (n = 7, 15d – 24d iNGNs) expressing ChR2(L132C) from AAV2 transductions. One circle represents one neuron, and red crosses show the average steady-state current density as the horizontal line and error bars denote SEM. (C) Representative current-clamp recordings of an iNGN (23d, left traces) and a rat hippocampal neuron (19 DIV HCN, right traces) expressing ChR2(L132C) by AAV transduction. Membrane potential was held at -75 mV and action potentials were driven by different frequencies of 20 pulses (1 ms duration) of 473 nm blue light, denoted by the vertical blue bars (not to scale, and shown only above the 5 Hz stimulation traces for clarity). The blue light power density was 70 mW/mm² for the iNGN and 36 mW/mm² for the HCN. The horizontal and vertical black scale bars denote 1000 ms and 80 mV, respectively. The numbers to the right of each firing trace are the number of action potentials.</p

Properties of spontaneous miniature excitatory postsynaptic currents (mEPSCs) in iNGNs.

<p>(A) Rise and decay kinetics of mEPSCs in 7d, 14d, 21d, 28d, 35d, and 42d iNGNs. Voltage-clamp recordings were done at -75 mV. Decay τ is plotted against rise time for each age and one circle represents one event. n is the number of cells recorded at each age. (B) Averaged event traces in 14d, 21d, 28d, 35d, and 42d iNGNs. n is the number of events recorded at each age.</p

Synapsin-I expression in iNGNs in long-term culture.

<p>Representative z-stack confocal microscope images of iNGNs are shown at 4, 7, 21, 35, 49, and 70 days, or of 21 DIV rat hippocampal neurons (HCN). Immunocytochemistry was used to observe MAP2 (green) and Synapsin-I (red) expression and colocalization in iNGNs, along with the nuclear stain DAPI (blue). Synapsin-I is expressed in iNGN dendrites from 4 to 70 days. Ages in days (d) are labeled on the left side of the images, and the white scale bar is 50 μm long.</p

SDF1-mediated CXCR4-internalization in NG108-15 cells.

<p><b>a.</b> Plot of cell capacitance (including stray capacitance) measured in whole cell configuration by patch-clamp technique against the membrane area. Cells were either not treated (red) or treated with 50 nM SDF1α (black) for >40 min. The membrane area was calculated from the measured cell diameter assuming a spherical shape of the cell. The specific cell capacitance (slope of the linear fit) decreases in presence of SDF1α. <b>b.</b> Confocal laser scanning micrographs of NG108-15 cells expressing CXCR4::eYFP in the presence of the inhibitor AMD3100 or the agonist SDF1α as indicated. While strong internalization is observed with SDF1α, only few vesicles are observed in presence of the inhibitor. <b>c.</b> Co-expression of CatCh::mKateA (red) and CXCR4::eYFP (cyan) in presence of 50 nM SDF1α. Note that after binding of SDF1α CXCR4 is internalized while CatCh mainly remains in the plasma membrane and is not directly affected by this growth factor (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165344#pone.0165344.s003" target="_blank">S3 Fig</a>). Scale bars represent 5 μm.</p

Confocal laser scanning micrographs of tCXCR4/CatCh protein in NG108-15 cells.

<p><b>a,b.</b> Z-stack overlay of a typical tCXCR4/CatCh cell either treated with 50 nM SDF1α for 45 min (a) or with AMD3100 (b). The tandem construct is internalized by the action of SDF1α (similar to CXCR4 shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165344#pone.0165344.g002" target="_blank">Fig 2</a>). <b>c.</b> Internalization of anti-CXCR4 antibody after immunoreaction with tCXCR4/CatCh in cells exposed to 50 nM SDF1α. The fluorescence signal of anti-CXCR4 (top, red) and tCXCR4/CatCh (middle, cyan) co-localize to a great extend as can be seen from white areas in the overlay (bottom). Scale bars represent 10 μm.</p