L5 Chrna2-Cre/<i>R26</i>^<i>tom</i> cells show Martinotti cell morphology and are low-threshold, slow accommodating firing.

<p>(A) Confocal image (20 μm, coronal slice) of primary auditory cortex of a <i>Chrna2-Cre/R26</i>^<i>tom</i> mouse showing tdTomato+ somas (red) in L5 with dense axonal arborizations in layer 1 (arrow in corner, scale bar = 100 μm). (B) Confocal image and tracing of a biocytin-filled tdTomato+ neuron (green). Reconstruction of soma and dendrites (black) and axon (red; scale bar = 20 μm) shows long axonal projections to layer 1. (C) Confocal images of a biocytin-filled (green) tdTomato+ neuron among several tdTomato+ neurons (red) show that cells have an ovoid cell body in L5, bipolar dendritic morphology, and proximal axonal arborizations. (D) Image illustrating how the <i>Chrna2-Cre/R26</i>^<i>tom</i> axons emerge from the main dendrite (circle). Scale bars = 50 μm. (E) Image showing the long axonal arborizations (arrows) from one biocytin-filled <i>Chrna2-Cre/R26</i>^<i>tom</i> cell (yellow) to layer 1 and the dense axonal ramifications (asterisk) in layer 1 from all <i>Chrna2-Cre/R26</i>^<i>tom</i> cells expressing tdTomato (red). Scale bars = 50 μm. (F) Example from another biocytin-filled <i>Chrna2-Cre/R26</i>^<i>tom</i> cell to emphasize axonal arborization extending laterally in layer 1, seen as a thin yellow axon at the border of the axonal plexus of <i>Chrna2-Cre/R26</i>^<i>tom</i> cell in layer 1. (G) <i>Top</i>: Example current clamp traces from a tdTomato+ cell showing low-threshold, accommodating firing (20 pA response in red, 100 pA in black, 500 ms) and rebound APs (−20 to −80 pA, 500 ms) typical for Martinotti cells. <i>Bottom</i>: Current clamp trace in response to a 200-pA, 1,000-ms-long stimulus used for analysis in (H). (H) <i>Left</i>: The frequency/current (f/I) curve of MCs^α2 shows an average firing rate around 20 Hz (at 200 pA, 1,000 ms) indicating slow spiking properties. <i>Middle</i>: Difference in maximum frequency and steady-state frequency for each neuron to a 200 pA, 1,000-ms-long current step highlights an accommodating discharge. The black line depicts the mean adaptation. <i>Right</i>: Spike-frequency adaptation is shown as a function of time. Data (<i>n</i> = 36 cells) are presented as mean ± standard error of the mean (SEM) and shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001392#pbio.2001392.s009" target="_blank">S1 Data</a>.</p

MC–PC inhibition is frequency dependent.

<p>(A) Expression of AAV-DIO-ChR2-EYFP (green) in MCs^α2 (red) in a primary auditory cortical slice used for optogenetic stimulation, with inset on L5 showing overlap of membrane expression in yellow (scale bars 50 μm). (B) Optogenetic activation (3-ms light pulses, 488 nm) of a group of MCs^α2 induced IPSPs in type A PCs (<i>left</i>) but not type B PCs (<i>right</i>) (<i>n</i> = 12 cells, single examples in grey, mean in black). (C) Example traces show MCs^α2 responses to blue light stimulation at various frequencies (3-ms blue light pulses at 2, 5, 15, 25, 40, and 70 Hz) and the corresponding IPSPs in a nearby type A PC (<i>n</i> = 12 cells, single examples in grey, mean in black). At higher frequencies (≥15Hz), the MC^α2–PC synapse showed depression. Note that MCs^α2 could not follow 70-Hz light stimulation for prolonged time. (D) <i>Top</i>: Continuous light stimulation of MCs^α2 (500 ms) generated large type A IPSP amplitudes (<i>middle</i>; <i>n</i> = 12 cells, single examples in grey, mean in black) similar in magnitude to IPSPs generated by high-frequency stimulation at 70 Hz. <i>Bottom</i>: Spike-frequency adaptation of MCs^α2 is shown as a function of time (see also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001392#pbio.2001392.s003" target="_blank">S3B–S3D Fig</a>). (E) Mean IPSP amplitudes in type A PCs following stimulation of MCs^α2 at different frequencies (from (C) and (D); 2 Hz: −1.57 ± 0.13 mV, 5 Hz: −1.70 ± 0.08 mV, 15 Hz: −3.05 ± 0.12 mV, 25 Hz: −3.20 ± 0.13 mV, 40 Hz: −3.76 ± 0.10 mV, 70 Hz: −4.37 ± 0.10 mV, 500 ms: −4.41 ± 0.07 mV; 2, 5 Hz versus 15, 25 Hz <i>p</i> < 0.0001; 15, 25 Hz versus 40 Hz <i>p</i> < 0.0001; 40 Hz versus 70 Hz, 500 ms <i>p</i> < 0.001; mean ± SEM, ANOVA, <i>n</i> = 12 cells, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001392#pbio.2001392.s011" target="_blank">S3 Data</a>).</p

MCs^α2 contribute to FDDI, and MC^α2 burst firing can reset type A PC spikes.

<p>(A) High-frequency stimulation (70 Hz, see arrow) of a presynaptic PC (▲) generates delayed IPSPs on a neighboring PC () via intermediate MCs^α2 (О). A mixed excitation (due to a monosynaptic PC–PC connection) followed by a disynaptic inhibition is shown (<i>n</i> = 12 cells, single examples in grey, mean in black). (B) An example of disynaptic inhibition alone (<i>top</i>) is shown (<i>n</i> = 12 cells, single examples in grey, mean in black). Silencing of HaloR-expressing MCs^α2 via green light (555 nm) prevents FDDI, although IPSPs are generated following termination of green light stimulation (<i>bottom</i>, <i>n</i> = 12 cells, single examples in grey, mean in black). (C) Mean IPSP amplitudes with (white) and without (green) FDDI at two different time points. (D) Responses from HaloR-expressing MCs^α2 (<i>top</i>) and local type A PCs (single-spiking and burst-spiking; <i>middle</i>) and type B PCs (<i>bottom</i>) are shown in presence of carbachol (10 μM). Green light stimulation (500 ms) hyperpolarizes HaloR-expressing MCs^α2 and upon termination MC^α2 rebound APs are triggered. This burst of APs generates robust inhibition in local postsynaptic type A PCs that synchronizes the timing of PC (rebound) APs. Kernel density estimates (orange) highlight increased (peaks) and decreased (valleys) co-occurance of APs. (E) Example of voltage clamp responses for type A (<i>top</i>) and type B (<i>bottom</i>) PCs in response to MCs^α2 burst firing (single examples in grey, mean in black). Values are shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001392#pbio.2001392.s012" target="_blank">S4 Data</a>.</p

Repeated bursts of MC^α2 inhibition synchronize type A PC spike trains via minimally depressing IPSPs.

<p>(A) Voltage traces from an unconnected pair of type A PCs (black and grey) with MCs^α2 stimulated in 15-Hz bursts at 2 Hz (blue dots). Orange rectangles highlight synchronous APs. (B) Mean cross-correlograms (<i>n</i> = 24 cells) show little synchrony before light stimulation and increased synchrony during light stimulation (15-Hz bursts in 2 Hz) of MCs^α2 as shown by a prominent peak around zero (and recurring peaks at every 500 ms). (C) Box plots of the synchrony indices for control and 15-Hz bursts show the significant increase of synchrony (0 no synchronization, 1 full synchronization) when MCs^α2 are stimulated by blue light in brief bursts (<i>n</i> = 12 dual recordings, <i>p</i> < 0.0001, two-tailed Student’s paired <i>t</i> test). Values are shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001392#pbio.2001392.s016" target="_blank">S8 Data</a>. (D) IPSPs in type A PCs (<i>n</i> = 24 cells, single examples in grey, mean in black) following burst protocol of 15-Hz stimulation in 2 Hz (top) and constant 15-Hz light stimulation (bottom). Note minimal-depressing inhibition in the top and the depression of IPSPs leading to a rapid diminution of inhibition in the bottom (red dashed lines for improved visualization).</p

MCs^α2 connect to local type A PCs but not type B PCs.

<p>(A) <i>Left</i>: The reconstruction of a typical type A PC showing a thick-tufted dendrite (scale bar = 40 μm) and its response to a 500-ms-long depolarizing (100 pA) and hyperpolarizing (−60 pA) stimulus. <i>Right</i>: A representative type B PC with a thin-tufted apical dendrite (scale bar = 40 μm) and its current clamp response (as for <i>left</i>). Note the deeper AHP (following a depolarizing current pulse), the more prominent sag (during a hyperpolarizing current pulse), as well as the pronounced rebound ADP (following a hyperpolarizing current pulse) in the type A PC compared to type B PC (see arrows). (B) Type A PCs can excite postsynaptic MCs^α2 (<i>inset</i>) and generate facilitating EPSPs (<i>left</i>, <i>n</i> = 7/9 pairs, 12 repetitions from one example pair are shown) when stimulated with high frequency (70 Hz), whereas type B PCs do not trigger EPSPs in MCs^α2 (<i>right</i>, <i>n</i> = 0/9 pairs, 12 repetitions). <i>Inset</i> shows experimental setup. (C) Typical MC^α2 discharges (<i>top</i>) to a 500-ms-long (25 pA) stimulus are shown. <i>Inset</i> shows experimental setup. MC^α2 spikes cause inhibition in postsynaptic type A PCs (<i>inset</i>) displaying synaptic depression (<i>middle left</i>, <i>n</i> = 7/9 pairs), whereas type B PCs do not receive MC^α2 inhibition (<i>middle right</i>, <i>n</i> = 0/9 pairs). Grey dashed lines highlight timing of presumably individually generated IPSPs for type A PCs, whereas for type B PCs, dashed line shows the lack of response. Example IPSP responses of 12 repetitions are shown in grey, mean response in black (<i>bottom</i>).</p

Type A PCs auto-synchronize via MC^α2 inhibition.

<p>(A) Coupling one PC (black) out of two unconnected type A PCs to the light source/optical feedback inhibition system shows unsynchronized activity before and synchronized APs during optical feedback inhibition. A total of 24 PC discharges pairwise aligned to the first PC AP with optical feedback inhibition are shown (<i>n</i> = 24 cells; 12 black and 12 grey spike trains). Kernel density estimates (orange) highlight increased (peaks) and decreased (valleys) co-occurrence of APs. Note that the time points of the blue light depend on the PC APs during the optical feedback inhibition and therefore vary between PC pairs. (B) <i>Top</i>: One pair of simultaneously recorded unconnected type A PCs (black and grey) showing discharges before and during the optical feedback inhibition. <i>Bottom</i>: Pairwise mutual information index versus time lag from recordings in (A) showing low mutual information for unconnected PCs and high mutual information around 0-ms lag for PCs coupled by optical feedback inhibition (<i>n</i> = 12 dual recordings, 24 cells). <i>Inset</i>: Amount of overlap in Venn diagrams (black and grey circles) shows low mutual information for unconnected (<i>left</i>) and significantly higher mutual information for coupled (<i>right</i>) PCs (<i>p</i> < 0.05, two-tailed Student’s paired <i>t</i> test). Values are shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001392#pbio.2001392.s017" target="_blank">S9 Data</a>.</p

Video_1_Direct Reprogramming of Adult Human Somatic Stem Cells Into Functional Neurons Using Sox2, Ascl1, and Neurog2.MOV

<p>Reprogramming of somatic cells into induced pluripotent stem cells (iPS) or directly into cells from a different lineage, including neurons, has revolutionized research in regenerative medicine in recent years. Mesenchymal stem cells are good candidates for lineage reprogramming and autologous transplantation, since they can be easily isolated from accessible sources in adult humans, such as bone marrow and dental tissues. Here, we demonstrate that expression of the transcription factors (TFs) SRY (sex determining region Y)-box 2 (Sox2), Mammalian achaete-scute homolog 1 (Ascl1), or Neurogenin 2 (Neurog2) is sufficient for reprogramming human umbilical cord mesenchymal stem cells (hUCMSC) into induced neurons (iNs). Furthermore, the combination of Sox2/Ascl1 or Sox2/Neurog2 is sufficient to reprogram up to 50% of transfected hUCMSCs into iNs showing electrical properties of mature neurons and establishing synaptic contacts with co-culture primary neurons. Finally, we show evidence supporting the notion that different combinations of TFs (Sox2/Ascl1 and Sox2/Neurog2) may induce multiple and overlapping neuronal phenotypes in lineage-reprogrammed iNs, suggesting that neuronal fate is determined by a combination of signals involving the TFs used for reprogramming but also the internal state of the converted cell. Altogether, the data presented here contribute to the advancement of techniques aiming at obtaining specific neuronal phenotypes from lineage-converted human somatic cells to treat neurological disorders.</p

Data_Sheet_1_Direct Reprogramming of Adult Human Somatic Stem Cells Into Functional Neurons Using Sox2, Ascl1, and Neurog2.DOCX

<p>Reprogramming of somatic cells into induced pluripotent stem cells (iPS) or directly into cells from a different lineage, including neurons, has revolutionized research in regenerative medicine in recent years. Mesenchymal stem cells are good candidates for lineage reprogramming and autologous transplantation, since they can be easily isolated from accessible sources in adult humans, such as bone marrow and dental tissues. Here, we demonstrate that expression of the transcription factors (TFs) SRY (sex determining region Y)-box 2 (Sox2), Mammalian achaete-scute homolog 1 (Ascl1), or Neurogenin 2 (Neurog2) is sufficient for reprogramming human umbilical cord mesenchymal stem cells (hUCMSC) into induced neurons (iNs). Furthermore, the combination of Sox2/Ascl1 or Sox2/Neurog2 is sufficient to reprogram up to 50% of transfected hUCMSCs into iNs showing electrical properties of mature neurons and establishing synaptic contacts with co-culture primary neurons. Finally, we show evidence supporting the notion that different combinations of TFs (Sox2/Ascl1 and Sox2/Neurog2) may induce multiple and overlapping neuronal phenotypes in lineage-reprogrammed iNs, suggesting that neuronal fate is determined by a combination of signals involving the TFs used for reprogramming but also the internal state of the converted cell. Altogether, the data presented here contribute to the advancement of techniques aiming at obtaining specific neuronal phenotypes from lineage-converted human somatic cells to treat neurological disorders.</p

Video_2_Direct Reprogramming of Adult Human Somatic Stem Cells Into Functional Neurons Using Sox2, Ascl1, and Neurog2.MOV

<p>Reprogramming of somatic cells into induced pluripotent stem cells (iPS) or directly into cells from a different lineage, including neurons, has revolutionized research in regenerative medicine in recent years. Mesenchymal stem cells are good candidates for lineage reprogramming and autologous transplantation, since they can be easily isolated from accessible sources in adult humans, such as bone marrow and dental tissues. Here, we demonstrate that expression of the transcription factors (TFs) SRY (sex determining region Y)-box 2 (Sox2), Mammalian achaete-scute homolog 1 (Ascl1), or Neurogenin 2 (Neurog2) is sufficient for reprogramming human umbilical cord mesenchymal stem cells (hUCMSC) into induced neurons (iNs). Furthermore, the combination of Sox2/Ascl1 or Sox2/Neurog2 is sufficient to reprogram up to 50% of transfected hUCMSCs into iNs showing electrical properties of mature neurons and establishing synaptic contacts with co-culture primary neurons. Finally, we show evidence supporting the notion that different combinations of TFs (Sox2/Ascl1 and Sox2/Neurog2) may induce multiple and overlapping neuronal phenotypes in lineage-reprogrammed iNs, suggesting that neuronal fate is determined by a combination of signals involving the TFs used for reprogramming but also the internal state of the converted cell. Altogether, the data presented here contribute to the advancement of techniques aiming at obtaining specific neuronal phenotypes from lineage-converted human somatic cells to treat neurological disorders.</p

Supplementary Material for: Thrombolysis for acute wake-up and unclear onset strokes with alteplase at 0.6 mg/kg in clinical practice: THAWS2 Study

Introduction: The aim of this study was to determine the safety and efficacy of intravenous (IV) alteplase at 0.6 mg/kg for patients with acute wake-up or unclear onset strokes in clinical practice. Methods: This multicenter observational study enrolled acute ischemic stroke patients with last-known-well time >4.5 h who had mismatch between DWI and FLAIR and were treated with IV alteplase. The safety outcomes were symptomatic intracranial hemorrhage (sICH) after thrombolysis, all-cause deaths and all adverse events. The efficacy outcomes were favorable outcome defined as an mRS score of 0–1 or recovery to the same mRS score as the premorbid score, complete independence defined as an mRS score of 0–1 at 90 days, and change in NIHSS at 24 h from baseline. Results: Sixty-six patients (35 females; mean age, 74±11 years; premorbid complete independence, 54 [82%]; median NIHSS on admission, 11) were enrolled at 15 hospitals. Two patients (3%) had sICH. Median NIHSS changed from 11 (IQR, 6.75–16.25) at baseline to 5 (3–12.25) at 24 h after alteplase initiation (change, –4.8±8.1). At discharge, 31 patients (47%) had favorable outcome and 29 (44%) had complete independence. None died within 90 days. Twenty-three (35%) also underwent mechanical thrombectomy (no sICH, NIHSS change of –8.5±7.3), of whom 11 (48%) were completely independent at discharge. Conclusions: In real-world clinical practice, IV alteplase for unclear onset stroke patients with DWI-FLAIR mismatch provided safe and efficacious outcomes comparable to those in previous trials. Additional mechanical thrombectomy was performed safely in them