Burst transmission in asymmetrical networks (DIV15).

<p>A—Explanatory diagrams for graphs presented on the left and right column respectively, with the same color code as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120680#pone.0120680.g004" target="_blank">Fig 4</a>. B—Typical example of a device showing unidirectional transmission. C—Results from a device where bursts also propagate in the reverse direction, albeit with a greater delay. Addition of 5 μM CNQX to the latter device yields the typical unidirectional transmission (D). Delays obtained by normalized cross correlation.</p

Compatibility of ChR2 and Calcium Orange.

<p>A- Quantification of background stimulation in ChR2(ET/TC) transduced cultures during calcium imaging with Calcium Orange at DIV12. The abscissa indicates the intensity of excitation light (549 nm) used during imaging relative to the recording conditions used in the rest of the experiments (1x); 2x is twice that level and 4x is 4 times that level. The different bursting rates at 2x and 4x were normalized by the bursting rates recorded at 1x for each culture so as to remove inter-culture variability for initial bursting rates. For information purpose, average absolute bursting rate at 1x was 5.9 ± 2.2 bursts per minute (n = 9 cultures). B- Absorption spectra of two ChR2 variants, including the ChR2(ET/TC) used in this study, as well as those of the commonly used Fluo-4 and its red-shifted counterpart Calcium Orange indicator. The figures of merit of each combination are indicated in % of maximal absorption.</p

Optogenetic induction of bursting events.

<p>A- Typical responses of a transduced culture (DIV 13) to different stimulation durations (1, 2, 4, 8, 16, 32, 64, 128, 256, 512 and 1024 ms). Vertical blue bars represent stimulation times and durations. No fluorescence data was recorded during the stimulations as the photodiode, recording directly the stimulated spot, was saturated. B- Same experiment as in A after addition of 10 μM CNQX (AMPA receptor antagonist).</p

Design of a simple neuronal device.

<p>A- Scheme showing the neuronal device featuring two micro-wells seeded with hippocampal neurons (DIV 15) and connected by an array of axon diodes. The neurons colored in green correspond to neurons that are transduced by ChR2. Neurons colored in purple are not transduced. B- Image of a neuronal device obtained by confocal fluorescence microscopy showing neurons expressing ChR2-YFP (false color).</p

Local stimulation and burst propagation (DIV15).

<p>A- Picture of a symmetrical two-compartments device with only one side transduced (left). Green and red channels are for ChR2-YFP and Calcium Orange indicator, respectively. This dual marking reveals that 70% of cells are expressing ChR2 inside the transduced compartment. B and C- Averaged response after stimulation of the transduced (B) and non-transduced (C) compartments. The solid colour lines correspond to the peristimulus fluorescence signal averaged over 30 stimulations (0.1 Hz in alternation) while the filled surfaces indicate associated standard deviations. Vertical dashed lines indicate stimulations. Diagrams are presented for clarity, with matching line and population colors. Green neurons are transduced neurons.</p

Early cortico-striatal synaptic disconnection after cortical fiber axotomy.

<p><b>a–f</b>: Cortical presynaptic structures (v-GLUT1, red) affixed to striatal dendrites (MAP-2, blue) in control conditions (<b>a</b>), three hours after axotomy (<b>b</b>)<i>scale bar: 20. </i><i>µm.</i> Note the disappearance of v-GLUT1 labeling suggesting cortical presynaptic degeneration. <b>c–f</b>: Representative high magnification images in control conditions (<b>c</b>) and two hours (<b>d</b>), four hours (<b>e</b>) and six hours (<b>f</b>) after axotomy. Note the fast cortico-striatal disconnection as soon as two hours after axotomy. <i>scale bar: 5. </i><i>µm. </i><b>g–n</b>: Live imaging of GFP-expressing striatal neurons at time 0h (<b>g,k</b>), 2h (<b>h,l</b>), 4h (<b>i,m</b>) and 6h (<b>j,n</b>) in control (<b>g–j</b>) or axotomy (<b>k–n</b>) conditions. Note the stability of striatal dendritic spines (arrow) in both conditions. s<i>cale bar: 15. </i><i>μm.</i></p

Pharmacological screen of synapto-protective drugs in 3C-microfluidic chip.

<p><b>a–j</b>: Representative high magnification images of cortical presynaptic structures (v-GLUT1, red) affixed to striatal dendrites (MAP-2, blue) in non-axotomized (<b>a–e</b>) and 3 hours post-axotomy (<b>f–j</b>) conditions, without treatment (<b>a,f</b>), or with resveratrol (20 µ<i>M </i><b>b,g</b>), 50 µ<i>M</i> z-VAD (<b>c,h</b>), 10 µ<i>M</i> Y27632 (<b>d,i</b>) or 5 m<i>M</i> NAD⁺ (<b>e,j</b>) pre-treatment. <i>scale bar: 5 </i><i>µm</i>. <b>k</b>: Quantification of cortical presynaptic structures affixed to MAP-2-positive striatal dendrites after cortical axotomy. The graph represents the relative number of synapses remaining compared to non-axotomized condition. Y27632 and NAD+ delay cortical synaptic loss whereas no significant protection is observed with resveratrol or z-VAD pre-treatment.(ANOVA 2, *p-value<0.05, **p-value<0.01). Synaptic degeneration was quantified using SynD software (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071103#pone.0071103.s001" target="_blank">Figure S1</a>) which allows automatic segmentation of striatal dendritic trees and co-localization of VGLUT1 positive synapses docked to the dendrites <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071103#pone.0071103-Schmitz1" target="_blank">[23]</a>.</p

Kinetics of axonal degeneration following cortical axotomy in a network.

<p><b>a,d</b>: Cortical, <b>b,e</b>: central and <b>c,f</b>: striatal, chambers of normal (<b>a–c</b>) or axotomized (<b>d–f</b>) reconstructed cortico-striatal networks (div14). Neurons were stained for MAP-2 (blue), α-tubulin (green) and v-GLUT1 (red). <b>a–c</b>: Cortical neurons send axons efficiently through the micro-channels and the central chamber to reach, and connect with, striatal neurons. <b>d–f</b>: Two hours after axotomy, no axons are present in the central chamber but axons inside the micro-channels and in the striatal chamber remain intact. <i>scale bar = 75 </i><i>µm </i><b>g–j</b>: Kinetics of degeneration of the distal part of the cortical axons following axotomy of cortical fibers in the receiving chamber. No axonal fragmentation (assessed by α-tubulin, green) is observed either in non-axotomized condition (<b>g</b>), or two hours after axotomy (<b>h</b>). The first tubulin beads (blebs) on cortical axons appear four hours after axotomy (<b>i</b>), and after six hours, all cortical axons disconnected from their soma are fragmented whereas striatal morphology remains intact (MAP-2, bleu) (<b>j</b>). <i>scale bar = 20 </i><i>µm.</i></p

Reconstruction of oriented neuronal networks in 3C-microfluidic chip.

<p><b>a</b>: Microfluidic neuronal culture devices are made up of two separate cell culture chambers (blue) interconnected by a series of asymmetrical micro-channels interrupted by a central narrow (50 µm-wide) channel that gives access to the central part of the axons. Each chamber is perfused individually by two reservoirs (indicated by R). <b>b</b>: Phase contrast image of a reconstructed oriented neuronal network in 3C-chip. Cortical neurons are seeded in the left chamber and connect striatal neurons seeded in the right chamber (red stars). The size of asymmetrical micro-channels allows the outgrowth of axons but prevents the entry of cell bodies; micro-channels allow the passage of axons only in a left-to-right direction (axonal diodes) <i>scale bar = 50 </i><i>µm</i>. <b>c–c’</b>: Immunofluorescent images of the receiving (right) chamber. Cortical axons exit micro-channels (green: α-tubulin) and cluster presynaptic markers (red: v-GLUT1) on striatal dendrites (blue: MAP-2). <b>c’</b>: Enlargement of the clustering of cortical presynaptic labeling on striatal dendrites, suggesting cortico-striatal synapse establishment. <i>scale bar = 20 μm.</i></p

ROCK inhibition lowers PrP^Sc level by rescuing TACE α-secretase neuroprotective activity towards PrP^C in a PDK1-dependent manner.

<p>(<b>A, B</b>) Amount of PrP^Sc in Rocki-Fk-1C11^5-HT cells differentiated into serotonergic neuronal cells for 4 days (4d) in the presence of Y-27632 (100 μM) or 22L-infected CGNs exposed for 6 h to Y-27632 (100 μM), as shown by western blotting (left) and quantified by ELISA (right). n = 10 for each condition. (<b>C</b>) Immunofluorescent labeling of TACE at the surface of Fk-infected 1C11^5-HT cells treated or not with the ROCK inhibitor (Y-27632, 100 μM) for 1 h. Scale bar, 50 μm. (<b>D</b>) Immunoblot analysis of sucrose gradient fractions of membranes of Fk-infected 1C11^5-HT cells treated or not with Y-27632 (100 μM, 1h) versus uninfected 1C11^5-HT to assess TACE displacement from the plasma membrane (FAK-enriched fractions) to caveolin-1-enriched vesicles. FAK = Focal Adhesion Kinase. Cav-1 = caveolin-1. (<b>E</b>) Immunofluorescent labeling of TACE at the surface of Rocki-Fk-1C11^5-HT differentiated for 4 days in the presence of Y-27632 (100 μM) versus uninfected 1C11^5-HT cells. Scale bar, 50 μm. (<b>F</b>) Western blot analysis (top) of the C1 fragment of PrP (C1) and full-length PrP (native) in Fk-infected 1C11^5-HT cells treated or not with Y-27632 (100 μM) or a combination of Y-27632 (100 μM) and TAPI-2 (100 μM) for 6 h versus uninfected cells and the ratio (bottom) of C1/native full-length PrP. n = 5 for each condition. (<b>G</b>) PDK1 activity in Fk-infected 1C11^5-HT cells treated or not with Y-27632 (100 μM, 1 h), in Fk-infected 1C11^5-HT bombarded with tungsten microprojectiles coated with ROCK-I or ROCK-II antibodies or both, or in Rocki-Fk-1C11^5-HT cells differentiated for 4 days in the presence of Y-27632 (100 μM) versus uninfected 1C11^5-HT cells. n = 5 for each condition. Values are the mean ± s.e.m. for all experiments. * <i>P</i> <0.01 versus uninfected cells. ** <i>P</i> <0.01 versus non-treated infected cells. # <i>P</i> <0.05 versus uninfected cells. ## <i>P</i> <0.05 versus non treated infected cells. ### <i>P</i> <0.05 versus infected cells treated with Y-27632.</p