Journal: Scientific Reports

Article Title: Optogenetic approaches addressing extracellular modulation of neural excitability

doi: 10.1038/srep23947

Figure Lengend Snippet: ( a ) Principle of the two-component optogenetic (TCO) approach. Upon illumination the light-activated proton pump may moderately acidify the local extracellular medium and activate acid-sensitive ion channels, ASICs, via their proton-sensing domain. This results in a remote but large sodium influx that can be used for sustained cell depolarization at moderate light intensities. In Xenopus oocytes, a light-driven proton pump of Coccomyxa subellipsoidea (CsR) was used. ( b–d ) Macroscopic currents of CsR T46N coexpressed with rat ASIC1a ( b ), rat ASIC2a ( c ) or rat ASIC3 ( d ) in oocytes at a molar RNA ratio of 1:1 (for ASIC3 of 2:1). Cells were illuminated with 560 nm light at different holding voltages at 0.1 mM MOPS and pH 7.5 under constant perfusion. The small outward directed pump currents (CsR) triggers large inward sodium currents (ASIC). Inset is a zoom-in to the initial pump activity directly after starting to illuminate CsR T46N -ASIC1a with green light. Note that ASIC1a and ASIC3 show strong inactivation in sustained light, whereas ASIC2a shows moderate to no inactivation at all.

Article Snippet: The protein sequence of rat ASIC2a (Genbank accession number AX286636) was human codon optimized and synthesized by Genscript. eArch3.0 and ASIC-YFP fusions were cloned into an AAV2 backbone either under a CaMKIIα or human synapsin promoter.

Techniques: Activity Assay

Journal: Scientific Reports

Article Title: Optogenetic approaches addressing extracellular modulation of neural excitability

doi: 10.1038/srep23947

Figure Lengend Snippet: ( a ) Current-voltage dependency of normalized photocurrents in 100 mM NaCl, 100 mM KCl or 100 mM CholineCl extracellular medium (all media contained additionally 1mM NaCl/KCl, 1 mM MgCl 2 , 0.1 mM CaCl 2 and 0.1 mM MOPS, pH 7.5, normalized to ASIC2a current activated by pH 4, mean +/− SD, n = 5). ( b ) ASIC2a currents measured during pH titration in darkness and comparison with photocurrents measured at pH 7.5 (mean +/− SD, n = 6). The green shaded region highlights the percent activation of ASIC2a by illumination with green light at 0.1 mM MOPS at −40 mV (data shown in ). Inset: representative pH activated current trace of ASIC2a at −40 mV. ( c ) Macroscopic currents of CsR T46N -ASIC2a activated by pH 4 or green light at different buffer concentrations (5 mM MOPS, 1 mM MOPS and 0.1 mM MOPS, −40 mV, constant perfusion). ( d ) Percent activation of ASIC2a by the light driven proton pump CsR T46N in different buffer concentrations (5 mM MOPS, 1 mM MOPS and 0.1 mM MOPS, −40 mV, n = 9, 100% activation taken as the peak ASIC current produced by pH 4, mean +/− SD, n = 9). ( e ) Normalized ASIC2a (red data points) and CsR T46N (green data points) photocurrents measured at different light intensities (0.1 mM MOPS, −40 mV, normalized to ASIC2a current activated by pH 4, mean +/− SD, n = 5). Inset: representative current traces at −40 mV.

Article Snippet: The protein sequence of rat ASIC2a (Genbank accession number AX286636) was human codon optimized and synthesized by Genscript. eArch3.0 and ASIC-YFP fusions were cloned into an AAV2 backbone either under a CaMKIIα or human synapsin promoter.

Techniques: Titration, Comparison, Activation Assay, Produced

Journal: Scientific Reports

Article Title: Optogenetic approaches addressing extracellular modulation of neural excitability

doi: 10.1038/srep23947

Figure Lengend Snippet: ( a ) Two-component Champ2.0 construct containing eArch (enhanced by trafficking sequence, TS) and ASIC2a, separated by a linker sequence and labeled with YFP. ( b ) Champ2.0 expression under CamKIIα or human synapsin (hSyn) promoters. ( c ) Representative voltage clamp trace for a Champ-expressing cell in response to a 1 s pulse of 560 nm light (green horizontal line). ( d ) Magnitude of outward (mean +/− SEM = 246 +/− 27 pA) and inward (−950 +/− 172 pA) components of the Champ current to a 1 s pulse of 560 nm light (n = 21). ( e ) Relationship between inward and outward components of the current (n = 21). Linear regression analysis yields R 2 = 0.33, p = 0.006 for difference of slope from zero (F(1, 19) = 9.447). ( f ) Example Champ responses to a 15 s light pulse in standard (25 mM HEPES, black trace) and weakly buffered (0.1 mM HEPES, grey trace) extracellular solution. ( g ) Peak inward currents in 25 and 0.1 mM HEPES for 1 s and 15 s light pulses. For 15 s light pulses, mean inward current is −164 pA +/−45 at 25 mM (n = 5) versus −379.9 +/−83 pA at 0.1 mM (n = 6), unpaired t-test with Welch’s correction: t = 2.278, df = 7.558, p = 0.0541). ( h ) Decay of inward current (final to peak current ratio for 15 s light pulse) in 25 mM (n = 5) versus 0.1 mM HEPES (n = 6), unpaired t-test with Welch’s correction: t = 4.779, df = 5.997, p = 0.0031. ( i ) Example membrane potential response to 1 s pulse of 560 nm light (green horizontal line) for a Champ expressing cell recorded in current clamp. ( j ) Magnitude of hyperpolarizing (eArch3.0-mediated, −33 +/− 3 mV) and depolarizing (ASIC2a-mediated, 87 +/− 6 mV) components of the light response (n = 13). ( k ) Relationship between Champ-mediated membrane hyperpolarization and depolarization (for 1 s light pulses). Linear regression analysis yields R 2 = 0.47, p = 0.0093 for difference of slope from zero (F(1, 11) = 9.885).

Article Snippet: The protein sequence of rat ASIC2a (Genbank accession number AX286636) was human codon optimized and synthesized by Genscript. eArch3.0 and ASIC-YFP fusions were cloned into an AAV2 backbone either under a CaMKIIα or human synapsin promoter.

Techniques: Construct, Sequencing, Labeling, Expressing, Membrane

Journal: Scientific Reports

Article Title: Optogenetic approaches addressing extracellular modulation of neural excitability

doi: 10.1038/srep23947

Figure Lengend Snippet: For each construct: a cartoon illustrates the structure of the two-component construct, confocal images demonstrate fluorescence expression in culture and graphs show the relative magnitude of the peak outward current and the current at the end of the light pulse. A more negative current at the end of the light pulse indicates a larger ASIC component. Insets: representative traces of the current responses to a 1 s pulse of 560 nm light for each two-component construct (timing of light pulse indicated by green horizontal line). All electrophysiological recordings were performed in low HEPES (0.1 mM) Tyrode’s solution. ( a ) eArch3.0-YFP only control (n = 9). ( b ) Co-transfection of eArch3.0 and ASIC2a: eArch3.0 is labeled with mCherry and ASIC2a is labeled with YFP to allow identification of both components in a single cell (n = 9). ( c ) Champ1.0: eArch3.0 and ASIC2a are separated during protein translation by the ribosomal skip sequence, p2A (n = 14). ( d ) Champ2.0: eArch3.0 and ASIC2a are fused by a 41 amino acid linker sequence (n = 17). ( e ) Champ3.0: eArch3.0 and ASIC2a are fused by a short linker sequence (23 amino acid membrane trafficking signal, TS) (n = 16).

Article Snippet: The protein sequence of rat ASIC2a (Genbank accession number AX286636) was human codon optimized and synthesized by Genscript. eArch3.0 and ASIC-YFP fusions were cloned into an AAV2 backbone either under a CaMKIIα or human synapsin promoter.

Techniques: Construct, Fluorescence, Expressing, Control, Cotransfection, Labeling, Sequencing, Membrane

Journal: Scientific Reports

Article Title: Optogenetic approaches addressing extracellular modulation of neural excitability

doi: 10.1038/srep23947

Figure Lengend Snippet: ( a , b ) ASIC1 ( a ) and ASIC2a ( b ) expression in CA1. ( c ) Change in membrane resistance during amiloride (ASIC antagonist) application (lilac-shaded region) (n = 6–18 cells, 9 mice, two-way ANOVA for interaction between time and drug treatment (F (12,314) = 2.617, p = 0.0018, asterisks indicate significant time points after Dunnet’s multiple comparison test). Grey data points: control experiment in which no amiloride was applied (n = 4–16, 6 mice) (no significant change from baseline). ( d ) Change in bystander current during amiloride application (F (12,314) = 2.473, p = 0.0032, asterisks indicate significant time points). No significant change from baseline for untreated control cells. ( e ) Examples bystander current at baseline baseline (dark red), after 20 mins amiloride (pink), and after 30 mins of washout (lilac). ( f ) Example ChR2 and eArch3.0 bystander currents in 500 μM acetazolamide (pale traces indicate baseline recordings, dark traces indicate 15 mins acetazolamide exposure). ( g ) Change in bystander current magnitude after acetazolamide application (thick line represents group mean) (ChR2, n = 7 cells, 3 mice, paired t-test: t = 1.313, df = 6, p = 0.2372; eArch3.0, n = 7 cells, 3 mice, paired t-test: t = 6.177, df = 6, p = 0.0008). ( h ) Occasionally, acetazolamide caused eArch3.0 bystander neurons to exhibit inward ASIC-like currents during green light. ( i ) Extracellular pH measurements in CA1 (contralateral to site of opsin injection) in acute slices using a solid state metal wire oxide pH sensor (100 μm diameter, 5X magnification). ( j ) pH change in response to three minutes of light stimulation (470 nm for ChR2 and YFP-controls, 560 nm for eArch3.0). For ChR2, n = 24 recording sites, 13 slices, 3 animals. For eArch3.0, n = 21 recording sites, 11 slices, 3 animals. For YFP controls, n = 22 recording sites, 12 slices, 2 animals. ( k ) Zoom-in of last minute of baseline pH recording and first two minutes of light stimulation.

Article Snippet: The protein sequence of rat ASIC2a (Genbank accession number AX286636) was human codon optimized and synthesized by Genscript. eArch3.0 and ASIC-YFP fusions were cloned into an AAV2 backbone either under a CaMKIIα or human synapsin promoter.

Techniques: Expressing, Membrane, Comparison, Control, Injection