whole-cell recordings Search Results


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  • 95
    Millipore whole cell voltage clamp recordings
    <t>Whole-cell</t> <t>voltage-clamp</t> <t>recordings</t> of tardbp Y220X/Y220X + MO larvae displayed a higher frequency of mEPC events at 2 dpf. A, 60 second sample traces from 10 minute recordings from the following 4 treatment groups: WT, WT + MO, tardbp Y220X/Y220X and tardbp Y220X/Y220X + MO. B, Overlay of all mEPC events recorded during a 10 minute recording in each treatment group. C, Scatterplots graphing exponential decay constant ( tau ) vs amplitude distributions for 5 individual recordings from each treatment group. Plotted mEPCs can be divided into two populations; slow decay tau ( tau > 4 ms), activity detected from neighbouring muscle fibers, and fast decay tau ( tau
    Whole Cell Voltage Clamp Recordings, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Olympus whole cell patch clamp recordings
    <t>Whole‐cell</t> targeting of Per1 ‐ EGFP +ve and EGFP ‐ve neurons in a living SCN slice. (A) Recording setup showing bright‐field image (4 ×) of a living coronal SCN slice (250 μm thick) taken at the mid‐rostro‐caudal level. (B) Image of Per1 ‐ EGFP +ve fluorescence signal (20 ×) taken from the slice shown in (A) with a FITC (488 nm, optimum for EGFP ) filter. Insets C1 and C2 are representative photomicrographs showing <t>patch</t> pipette targeting of EGFP −ve ( C1 , follow vertically) or Per1 ‐ EGFP +ve ( C2 ) neurons (40 ×) in the slice shown in A and B. Insets rows show bright‐field, fluorescence ( FITC ), and merged bright‐field and fluorescence images. (D and E) Spontaneous membrane excitability states of Per1 ‐ EGFP +ve (D) and EGFP −ve (E) SCN neurons over the day. (D) Example of typical <t>current‐clamp</t> traces from Per1 ‐ EGFP +ve SCN neurons recorded in the morning ( ZT 2–5) and afternoon ( ZT 6–10). Note that Per1 ‐ EGFP +ve neurons recorded in the morning are at moderate resting membrane potential ( RMP : ~ −45 mV ) and discharging action potentials ( AP s), while in the afternoon they become hyperexcited with RMP s of −25 to −35 mV and unable to generate AP s. These cells are either silent or generating depolarized low‐amplitude membrane oscillations ( DLAMO s). By contrast, EGFP −ve neurons (E) remain at moderate RMP throughout the day, firing AP s. Dashed lines in C indicate the outline of the patch pipette. SCN , suprachiasmatic nuclei; OX , optic chiasm; P , patch pipette. [Colour figure can be viewed at wileyonlinelibrary.com ].
    Whole Cell Patch Clamp Recordings, supplied by Olympus, used in various techniques. Bioz Stars score: 94/100, based on 691 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    HEKA Elektronik whole cell patch clamp recordings
    Effects of μ-TRTX-Ca2a on Na v 1.2–Na v 1.9 channels. (A–H) Representative Na v 1.2–Na v 1.9 current traces before (black) and after (red) addition of Ca2a. Ca2a at 1 μM inhibited Na v 1.2–Na v 1.3, Na v 1.6, and Na v 1.7. 10 μM Ca2a showed no obvious effect on Na v 1.4–Na v 1.5 or Na v 1.8–Na v 1.9 current. Inset above panel (A) shows the pulse protocol for recording Na v 1.2–Na v 1.7 channel currents. Inset above panel (G) shows the pulse protocol for recording Na v 1.8 channel current. Inset above panel (H) shows the pulse protocol for recording Na v 1.9 channel current. (I) Concentration-response curves of Ca2a at Na v 1.2–Na v 1.3, Na v 1.6, and Na v 1.7 assessed by <t>whole-cell</t> <t>patch-clamp</t> experiments. Data are mean ± SEM, with n = 4–7 cells per data point.
    Whole Cell Patch Clamp Recordings, supplied by HEKA Elektronik, used in various techniques. Bioz Stars score: 93/100, based on 410 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Sutter Instrument whole cell patch clamp recordings
    Ca 2+ dependence of anion selectivity for TMEM16A is abolished by mutations of basic residues. ( A ) Currents recorded from voltage ramps from −80 to +80 mV in WT TMEM16A in <t>whole-cell</t> <t>patch</t> <t>clamp</t> with 400 nM Ca 2+ in the pipette. Intracellular solution contained 140 mM NaCl, whereas extracellular ion concentrations are as indicated. ( B ) Currents recorded from voltage ramps from −80 to +80 mV in WT TMEM16A with 1 mMCa 2+ in the pipette. ( C ) Permeability ratios are determined by solving the Goldman–Hodgkin–Katz voltage equation for each solution compared with chloride in the indicated concentrations of Ca 2+ . Permeability ratios for thiocyanate over chloride are shown for alanine mutants at ( D ) 400 nM Ca 2+ and ( E ) 1 mM Ca 2+ . N.S., not significant. *** P
    Whole Cell Patch Clamp Recordings, supplied by Sutter Instrument, used in various techniques. Bioz Stars score: 92/100, based on 193 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Warner Instruments two microelectrode voltage clamp technique
    A comparison of normalized peak current amplitudes for wild-type and mutant hNa v 1.5 channels expressed by <t>two</t> different expression systems. Whole-cell currents were recorded by the patch <t>clamp</t> <t>technique</t> (HEK293 cells) and by the <t>two-microelectrode</t> <t>voltage</t> clamp technique (oocytes) as described in “ Methods ”. L212P and T220I exhibited similar relative expression levels in both systems. With E161K, D1275N, and P1298L, whole-cell currents, normalized to hNa v 1.5 values, were significantly smaller for the oocyte system (* indicates p
    Two Microelectrode Voltage Clamp Technique, supplied by Warner Instruments, used in various techniques. Bioz Stars score: 89/100, based on 54 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Olympus whole cell voltage clamp recordings
    Insulin induced a slight hyperpolarization in DMV neurons and significantly decreased <t>whole</t> <t>cell</t> input resistance in neurons that hyperpolarized. A : sample traces of <t>current-clamp</t> <t>recordings</t> from the same unidentified DMV neuron in aCSF + TTX (2 μM)
    Whole Cell Voltage Clamp Recordings, supplied by Olympus, used in various techniques. Bioz Stars score: 92/100, based on 146 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Warner Instruments whole cell patch clamp recordings
    Ex vivo — in vitro <t>whole-cell</t> <t>patch</t> <t>recordings</t> of Cy3- Wisteria floribunda agglutinin (WFA)-labeled fast-spiking interneurons (FSIs) after transcranial magnetic stimulation (TMS). (A) Drawing showing placement and orientation of the figure-of-eight coil used to apply the intermittent theta-burst (iTBS) protocol (see B ) to the rat brain. At the orientation shown, a mediolaterally oriented electric field is induced within the brain, optimally suited to depolarize axons of the corpus callosum. (C) Red fluorescent Cy3-WFA-labeled perineuronal net of a cortical FSIs revealed by fluorescence microscopy. (D) The same neuron shown in infrared phase contrast illumination. (E) Same as in (D) but with the patch pipette attached to the cell. (F) Cell filled with Alexa488.
    Whole Cell Patch Clamp Recordings, supplied by Warner Instruments, used in various techniques. Bioz Stars score: 92/100, based on 65 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Warner Instruments two electrode voltage clamp techniques
    Cellular pH and Chloride Transport Studies (A) ClC-7 p.Tyr715Cys expression causes increased current in Xenopus oocytes. <t>Two-electrode</t> <t>voltage-clamp</t> current traces are shown for uninjected (black), WT ClC-7 PM (blue), and p.Tyr715Cys ClC-7 PM (red) in response to a series of 1 s voltage steps from −100 mV to + 80 mV in increments of 20 mV, followed by a step to −80 mV for 1.4 s before return to a holding potential of −30 mV. (B) Bar graphs of current (+/− SEM, measured at +80 mV) or surface expression of uninjected (black), WT ClC-7 PM , or p.Tyr715Cys ClC-7 PM . For current measurements, n = 3 (uninjected), 8 (WT), or 6 (p.Tyr715Cys) from a single batch of oocytes; the experiment was reproduced 11 times in separate oocyte batches with essentially identical results. For surface expression, five separate surface biotinylation experiments were performed (with 20 each of WT and p.Tyr715Cys oocytes); intensities were normalized by the WT band intensity and averaged. The currents shown in (A) were from one of the same batches of oocytes used for the surface-expression experiments. (C) Lysosomal pH in control and proband fibroblasts was measured with OG488 ratiometric imaging. Raw fluorescence ratios are shown for individual cells (pale circles) and averages (+/− SEM) of n = ∼50 cells (dark circles) from two independent experiments each on control neonatal fibroblasts (1.54 ± 0.1) or primary fibroblasts from either proband (proband 1, 1.30 ± 0.07; proband 2, 1.28 ± 0.08). Ratios from bafilomycin-treated cells from each are also shown as a negative control. The lower the 490/440nm fluorescence ratio, the lower the pH. An image of an OG488-treated proband cell is shown in the inset. ). Error bars indicate SEM; p values as shown. (E) Cultured fibroblasts incubated with LysoTracker Red DND-99. Fluorescent intensity was significantly greater in cells of probands 1 and 2 than in control cells. Nuclei are stained with Hoechst (blue channel). (F) Mean (+/− SEM) integrated intensity of LysoTracker Red DND-99 (red channel) per cell is plotted in relative fluorescence units (RFU); values are 10 7 . Nuclei are stained with Hoechst (blue channel). Fluorescent intensity was significantly greater in cells of probands 1 and 2 than in control cells. Abbreviations are as follows: SEM, standard error the mean; WT, wild-type.
    Two Electrode Voltage Clamp Techniques, supplied by Warner Instruments, used in various techniques. Bioz Stars score: 88/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    HEKA Electronics Incorporated whole cell patch clamp recording
    No changes in excitatory synaptic and intrinsic properties in Shank2 −/− Purkinje cells ex vivo . ( a ) <t>Recording</t> configuration for <t>voltage-clamp</t> recordings of PF–PC synaptic transmission. Inset: an example PF-EPSC. ( b – e ) With comparable holding current (at −65 mV) ( P =1) and input resistance (Ri) ( P =0.8), PC EPSC rise time ( P =0.2) and EPSC decay time ( P =0.3) are not different between WT ( n =9/6, cells per animals) and Shank2 −/− ( n =7/6). ( f ) Example EPSCs in response to 3, 6, 9, 12 and 15 μA stimulation. ( g , h ) Varying stimulation strength ( P =0.9, repeated-measures ANOVA) and inter-stimulus interval ( P =0.2, repeated-measures ANOVA) evoked comparable EPSC amplitude or facilitation (WT, n =11/3; Shank2 −/− 15/3). ( i ) Recording configuration for <t>whole-cell</t> recording. Inset: an example action potential. ( j – m ) Action potential threshold ( P =0.8), amplitude ( P =0.1), half-width ( P =0.7) and after-hyperpolarization ( P =0.2) were not different (WT, n =10/6; Shank2 −/− , n =11/6). ( n ) Example traces of intrinsic Purkinje cell excitability as apparent from action potential firing evoked by 300 pA current injections. ( o ) No difference in evoked firing frequency relative to various levels of current injections (WT, n =10/5; Shank2 −/− , n =11/5, P =0.1, repeated-measures ANOVA). Inset barplot shows average slope of firing rate per current step ( P =1). Data are represented as mean±s.e.m. Two-sided t -tests were used, unless stated otherwise.
    Whole Cell Patch Clamp Recording, supplied by HEKA Electronics Incorporated, used in various techniques. Bioz Stars score: 90/100, based on 41 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Harvard Bioscience whole cell recording
    No changes in excitatory synaptic and intrinsic properties in Shank2 −/− Purkinje cells ex vivo . ( a ) <t>Recording</t> configuration for <t>voltage-clamp</t> recordings of PF–PC synaptic transmission. Inset: an example PF-EPSC. ( b – e ) With comparable holding current (at −65 mV) ( P =1) and input resistance (Ri) ( P =0.8), PC EPSC rise time ( P =0.2) and EPSC decay time ( P =0.3) are not different between WT ( n =9/6, cells per animals) and Shank2 −/− ( n =7/6). ( f ) Example EPSCs in response to 3, 6, 9, 12 and 15 μA stimulation. ( g , h ) Varying stimulation strength ( P =0.9, repeated-measures ANOVA) and inter-stimulus interval ( P =0.2, repeated-measures ANOVA) evoked comparable EPSC amplitude or facilitation (WT, n =11/3; Shank2 −/− 15/3). ( i ) Recording configuration for <t>whole-cell</t> recording. Inset: an example action potential. ( j – m ) Action potential threshold ( P =0.8), amplitude ( P =0.1), half-width ( P =0.7) and after-hyperpolarization ( P =0.2) were not different (WT, n =10/6; Shank2 −/− , n =11/6). ( n ) Example traces of intrinsic Purkinje cell excitability as apparent from action potential firing evoked by 300 pA current injections. ( o ) No difference in evoked firing frequency relative to various levels of current injections (WT, n =10/5; Shank2 −/− , n =11/5, P =0.1, repeated-measures ANOVA). Inset barplot shows average slope of firing rate per current step ( P =1). Data are represented as mean±s.e.m. Two-sided t -tests were used, unless stated otherwise.
    Whole Cell Recording, supplied by Harvard Bioscience, used in various techniques. Bioz Stars score: 91/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Narishige whole cell recording
    No changes in excitatory synaptic and intrinsic properties in Shank2 −/− Purkinje cells ex vivo . ( a ) <t>Recording</t> configuration for <t>voltage-clamp</t> recordings of PF–PC synaptic transmission. Inset: an example PF-EPSC. ( b – e ) With comparable holding current (at −65 mV) ( P =1) and input resistance (Ri) ( P =0.8), PC EPSC rise time ( P =0.2) and EPSC decay time ( P =0.3) are not different between WT ( n =9/6, cells per animals) and Shank2 −/− ( n =7/6). ( f ) Example EPSCs in response to 3, 6, 9, 12 and 15 μA stimulation. ( g , h ) Varying stimulation strength ( P =0.9, repeated-measures ANOVA) and inter-stimulus interval ( P =0.2, repeated-measures ANOVA) evoked comparable EPSC amplitude or facilitation (WT, n =11/3; Shank2 −/− 15/3). ( i ) Recording configuration for <t>whole-cell</t> recording. Inset: an example action potential. ( j – m ) Action potential threshold ( P =0.8), amplitude ( P =0.1), half-width ( P =0.7) and after-hyperpolarization ( P =0.2) were not different (WT, n =10/6; Shank2 −/− , n =11/6). ( n ) Example traces of intrinsic Purkinje cell excitability as apparent from action potential firing evoked by 300 pA current injections. ( o ) No difference in evoked firing frequency relative to various levels of current injections (WT, n =10/5; Shank2 −/− , n =11/5, P =0.1, repeated-measures ANOVA). Inset barplot shows average slope of firing rate per current step ( P =1). Data are represented as mean±s.e.m. Two-sided t -tests were used, unless stated otherwise.
    Whole Cell Recording, supplied by Narishige, used in various techniques. Bioz Stars score: 92/100, based on 39 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Olympus whole cell recordings
    No changes in excitatory synaptic and intrinsic properties in Shank2 −/− Purkinje cells ex vivo . ( a ) <t>Recording</t> configuration for <t>voltage-clamp</t> recordings of PF–PC synaptic transmission. Inset: an example PF-EPSC. ( b – e ) With comparable holding current (at −65 mV) ( P =1) and input resistance (Ri) ( P =0.8), PC EPSC rise time ( P =0.2) and EPSC decay time ( P =0.3) are not different between WT ( n =9/6, cells per animals) and Shank2 −/− ( n =7/6). ( f ) Example EPSCs in response to 3, 6, 9, 12 and 15 μA stimulation. ( g , h ) Varying stimulation strength ( P =0.9, repeated-measures ANOVA) and inter-stimulus interval ( P =0.2, repeated-measures ANOVA) evoked comparable EPSC amplitude or facilitation (WT, n =11/3; Shank2 −/− 15/3). ( i ) Recording configuration for <t>whole-cell</t> recording. Inset: an example action potential. ( j – m ) Action potential threshold ( P =0.8), amplitude ( P =0.1), half-width ( P =0.7) and after-hyperpolarization ( P =0.2) were not different (WT, n =10/6; Shank2 −/− , n =11/6). ( n ) Example traces of intrinsic Purkinje cell excitability as apparent from action potential firing evoked by 300 pA current injections. ( o ) No difference in evoked firing frequency relative to various levels of current injections (WT, n =10/5; Shank2 −/− , n =11/5, P =0.1, repeated-measures ANOVA). Inset barplot shows average slope of firing rate per current step ( P =1). Data are represented as mean±s.e.m. Two-sided t -tests were used, unless stated otherwise.
    Whole Cell Recordings, supplied by Olympus, used in various techniques. Bioz Stars score: 93/100, based on 474 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    dagan corp two microelectrode voltage clamp technique
    No changes in excitatory synaptic and intrinsic properties in Shank2 −/− Purkinje cells ex vivo . ( a ) <t>Recording</t> configuration for <t>voltage-clamp</t> recordings of PF–PC synaptic transmission. Inset: an example PF-EPSC. ( b – e ) With comparable holding current (at −65 mV) ( P =1) and input resistance (Ri) ( P =0.8), PC EPSC rise time ( P =0.2) and EPSC decay time ( P =0.3) are not different between WT ( n =9/6, cells per animals) and Shank2 −/− ( n =7/6). ( f ) Example EPSCs in response to 3, 6, 9, 12 and 15 μA stimulation. ( g , h ) Varying stimulation strength ( P =0.9, repeated-measures ANOVA) and inter-stimulus interval ( P =0.2, repeated-measures ANOVA) evoked comparable EPSC amplitude or facilitation (WT, n =11/3; Shank2 −/− 15/3). ( i ) Recording configuration for <t>whole-cell</t> recording. Inset: an example action potential. ( j – m ) Action potential threshold ( P =0.8), amplitude ( P =0.1), half-width ( P =0.7) and after-hyperpolarization ( P =0.2) were not different (WT, n =10/6; Shank2 −/− , n =11/6). ( n ) Example traces of intrinsic Purkinje cell excitability as apparent from action potential firing evoked by 300 pA current injections. ( o ) No difference in evoked firing frequency relative to various levels of current injections (WT, n =10/5; Shank2 −/− , n =11/5, P =0.1, repeated-measures ANOVA). Inset barplot shows average slope of firing rate per current step ( P =1). Data are represented as mean±s.e.m. Two-sided t -tests were used, unless stated otherwise.
    Two Microelectrode Voltage Clamp Technique, supplied by dagan corp, used in various techniques. Bioz Stars score: 89/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Carl Zeiss whole cell recording
    No changes in excitatory synaptic and intrinsic properties in Shank2 −/− Purkinje cells ex vivo . ( a ) <t>Recording</t> configuration for <t>voltage-clamp</t> recordings of PF–PC synaptic transmission. Inset: an example PF-EPSC. ( b – e ) With comparable holding current (at −65 mV) ( P =1) and input resistance (Ri) ( P =0.8), PC EPSC rise time ( P =0.2) and EPSC decay time ( P =0.3) are not different between WT ( n =9/6, cells per animals) and Shank2 −/− ( n =7/6). ( f ) Example EPSCs in response to 3, 6, 9, 12 and 15 μA stimulation. ( g , h ) Varying stimulation strength ( P =0.9, repeated-measures ANOVA) and inter-stimulus interval ( P =0.2, repeated-measures ANOVA) evoked comparable EPSC amplitude or facilitation (WT, n =11/3; Shank2 −/− 15/3). ( i ) Recording configuration for <t>whole-cell</t> recording. Inset: an example action potential. ( j – m ) Action potential threshold ( P =0.8), amplitude ( P =0.1), half-width ( P =0.7) and after-hyperpolarization ( P =0.2) were not different (WT, n =10/6; Shank2 −/− , n =11/6). ( n ) Example traces of intrinsic Purkinje cell excitability as apparent from action potential firing evoked by 300 pA current injections. ( o ) No difference in evoked firing frequency relative to various levels of current injections (WT, n =10/5; Shank2 −/− , n =11/5, P =0.1, repeated-measures ANOVA). Inset barplot shows average slope of firing rate per current step ( P =1). Data are represented as mean±s.e.m. Two-sided t -tests were used, unless stated otherwise.
    Whole Cell Recording, supplied by Carl Zeiss, used in various techniques. Bioz Stars score: 91/100, based on 38 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Sutter Instrument whole cell recordings
    No changes in excitatory synaptic and intrinsic properties in Shank2 −/− Purkinje cells ex vivo . ( a ) <t>Recording</t> configuration for <t>voltage-clamp</t> recordings of PF–PC synaptic transmission. Inset: an example PF-EPSC. ( b – e ) With comparable holding current (at −65 mV) ( P =1) and input resistance (Ri) ( P =0.8), PC EPSC rise time ( P =0.2) and EPSC decay time ( P =0.3) are not different between WT ( n =9/6, cells per animals) and Shank2 −/− ( n =7/6). ( f ) Example EPSCs in response to 3, 6, 9, 12 and 15 μA stimulation. ( g , h ) Varying stimulation strength ( P =0.9, repeated-measures ANOVA) and inter-stimulus interval ( P =0.2, repeated-measures ANOVA) evoked comparable EPSC amplitude or facilitation (WT, n =11/3; Shank2 −/− 15/3). ( i ) Recording configuration for <t>whole-cell</t> recording. Inset: an example action potential. ( j – m ) Action potential threshold ( P =0.8), amplitude ( P =0.1), half-width ( P =0.7) and after-hyperpolarization ( P =0.2) were not different (WT, n =10/6; Shank2 −/− , n =11/6). ( n ) Example traces of intrinsic Purkinje cell excitability as apparent from action potential firing evoked by 300 pA current injections. ( o ) No difference in evoked firing frequency relative to various levels of current injections (WT, n =10/5; Shank2 −/− , n =11/5, P =0.1, repeated-measures ANOVA). Inset barplot shows average slope of firing rate per current step ( P =1). Data are represented as mean±s.e.m. Two-sided t -tests were used, unless stated otherwise.
    Whole Cell Recordings, supplied by Sutter Instrument, used in various techniques. Bioz Stars score: 92/100, based on 352 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Millipore whole cell patch clamp recordings
    No changes in excitatory synaptic and intrinsic properties in Shank2 −/− Purkinje cells ex vivo . ( a ) <t>Recording</t> configuration for <t>voltage-clamp</t> recordings of PF–PC synaptic transmission. Inset: an example PF-EPSC. ( b – e ) With comparable holding current (at −65 mV) ( P =1) and input resistance (Ri) ( P =0.8), PC EPSC rise time ( P =0.2) and EPSC decay time ( P =0.3) are not different between WT ( n =9/6, cells per animals) and Shank2 −/− ( n =7/6). ( f ) Example EPSCs in response to 3, 6, 9, 12 and 15 μA stimulation. ( g , h ) Varying stimulation strength ( P =0.9, repeated-measures ANOVA) and inter-stimulus interval ( P =0.2, repeated-measures ANOVA) evoked comparable EPSC amplitude or facilitation (WT, n =11/3; Shank2 −/− 15/3). ( i ) Recording configuration for <t>whole-cell</t> recording. Inset: an example action potential. ( j – m ) Action potential threshold ( P =0.8), amplitude ( P =0.1), half-width ( P =0.7) and after-hyperpolarization ( P =0.2) were not different (WT, n =10/6; Shank2 −/− , n =11/6). ( n ) Example traces of intrinsic Purkinje cell excitability as apparent from action potential firing evoked by 300 pA current injections. ( o ) No difference in evoked firing frequency relative to various levels of current injections (WT, n =10/5; Shank2 −/− , n =11/5, P =0.1, repeated-measures ANOVA). Inset barplot shows average slope of firing rate per current step ( P =1). Data are represented as mean±s.e.m. Two-sided t -tests were used, unless stated otherwise.
    Whole Cell Patch Clamp Recordings, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 67 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Warner Instruments whole cell recordings
    No changes in excitatory synaptic and intrinsic properties in Shank2 −/− Purkinje cells ex vivo . ( a ) <t>Recording</t> configuration for <t>voltage-clamp</t> recordings of PF–PC synaptic transmission. Inset: an example PF-EPSC. ( b – e ) With comparable holding current (at −65 mV) ( P =1) and input resistance (Ri) ( P =0.8), PC EPSC rise time ( P =0.2) and EPSC decay time ( P =0.3) are not different between WT ( n =9/6, cells per animals) and Shank2 −/− ( n =7/6). ( f ) Example EPSCs in response to 3, 6, 9, 12 and 15 μA stimulation. ( g , h ) Varying stimulation strength ( P =0.9, repeated-measures ANOVA) and inter-stimulus interval ( P =0.2, repeated-measures ANOVA) evoked comparable EPSC amplitude or facilitation (WT, n =11/3; Shank2 −/− 15/3). ( i ) Recording configuration for <t>whole-cell</t> recording. Inset: an example action potential. ( j – m ) Action potential threshold ( P =0.8), amplitude ( P =0.1), half-width ( P =0.7) and after-hyperpolarization ( P =0.2) were not different (WT, n =10/6; Shank2 −/− , n =11/6). ( n ) Example traces of intrinsic Purkinje cell excitability as apparent from action potential firing evoked by 300 pA current injections. ( o ) No difference in evoked firing frequency relative to various levels of current injections (WT, n =10/5; Shank2 −/− , n =11/5, P =0.1, repeated-measures ANOVA). Inset barplot shows average slope of firing rate per current step ( P =1). Data are represented as mean±s.e.m. Two-sided t -tests were used, unless stated otherwise.
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    Warner Instruments whole cell patch clamp recordings borosilicate glass electrodes
    No changes in excitatory synaptic and intrinsic properties in Shank2 −/− Purkinje cells ex vivo . ( a ) <t>Recording</t> configuration for <t>voltage-clamp</t> recordings of PF–PC synaptic transmission. Inset: an example PF-EPSC. ( b – e ) With comparable holding current (at −65 mV) ( P =1) and input resistance (Ri) ( P =0.8), PC EPSC rise time ( P =0.2) and EPSC decay time ( P =0.3) are not different between WT ( n =9/6, cells per animals) and Shank2 −/− ( n =7/6). ( f ) Example EPSCs in response to 3, 6, 9, 12 and 15 μA stimulation. ( g , h ) Varying stimulation strength ( P =0.9, repeated-measures ANOVA) and inter-stimulus interval ( P =0.2, repeated-measures ANOVA) evoked comparable EPSC amplitude or facilitation (WT, n =11/3; Shank2 −/− 15/3). ( i ) Recording configuration for <t>whole-cell</t> recording. Inset: an example action potential. ( j – m ) Action potential threshold ( P =0.8), amplitude ( P =0.1), half-width ( P =0.7) and after-hyperpolarization ( P =0.2) were not different (WT, n =10/6; Shank2 −/− , n =11/6). ( n ) Example traces of intrinsic Purkinje cell excitability as apparent from action potential firing evoked by 300 pA current injections. ( o ) No difference in evoked firing frequency relative to various levels of current injections (WT, n =10/5; Shank2 −/− , n =11/5, P =0.1, repeated-measures ANOVA). Inset barplot shows average slope of firing rate per current step ( P =1). Data are represented as mean±s.e.m. Two-sided t -tests were used, unless stated otherwise.
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    Carl Zeiss whole cell patch recordings
    No changes in excitatory synaptic and intrinsic properties in Shank2 −/− Purkinje cells ex vivo . ( a ) <t>Recording</t> configuration for <t>voltage-clamp</t> recordings of PF–PC synaptic transmission. Inset: an example PF-EPSC. ( b – e ) With comparable holding current (at −65 mV) ( P =1) and input resistance (Ri) ( P =0.8), PC EPSC rise time ( P =0.2) and EPSC decay time ( P =0.3) are not different between WT ( n =9/6, cells per animals) and Shank2 −/− ( n =7/6). ( f ) Example EPSCs in response to 3, 6, 9, 12 and 15 μA stimulation. ( g , h ) Varying stimulation strength ( P =0.9, repeated-measures ANOVA) and inter-stimulus interval ( P =0.2, repeated-measures ANOVA) evoked comparable EPSC amplitude or facilitation (WT, n =11/3; Shank2 −/− 15/3). ( i ) Recording configuration for <t>whole-cell</t> recording. Inset: an example action potential. ( j – m ) Action potential threshold ( P =0.8), amplitude ( P =0.1), half-width ( P =0.7) and after-hyperpolarization ( P =0.2) were not different (WT, n =10/6; Shank2 −/− , n =11/6). ( n ) Example traces of intrinsic Purkinje cell excitability as apparent from action potential firing evoked by 300 pA current injections. ( o ) No difference in evoked firing frequency relative to various levels of current injections (WT, n =10/5; Shank2 −/− , n =11/5, P =0.1, repeated-measures ANOVA). Inset barplot shows average slope of firing rate per current step ( P =1). Data are represented as mean±s.e.m. Two-sided t -tests were used, unless stated otherwise.
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    dagan corp two electrode voltage clamp technique
    No changes in excitatory synaptic and intrinsic properties in Shank2 −/− Purkinje cells ex vivo . ( a ) <t>Recording</t> configuration for <t>voltage-clamp</t> recordings of PF–PC synaptic transmission. Inset: an example PF-EPSC. ( b – e ) With comparable holding current (at −65 mV) ( P =1) and input resistance (Ri) ( P =0.8), PC EPSC rise time ( P =0.2) and EPSC decay time ( P =0.3) are not different between WT ( n =9/6, cells per animals) and Shank2 −/− ( n =7/6). ( f ) Example EPSCs in response to 3, 6, 9, 12 and 15 μA stimulation. ( g , h ) Varying stimulation strength ( P =0.9, repeated-measures ANOVA) and inter-stimulus interval ( P =0.2, repeated-measures ANOVA) evoked comparable EPSC amplitude or facilitation (WT, n =11/3; Shank2 −/− 15/3). ( i ) Recording configuration for <t>whole-cell</t> recording. Inset: an example action potential. ( j – m ) Action potential threshold ( P =0.8), amplitude ( P =0.1), half-width ( P =0.7) and after-hyperpolarization ( P =0.2) were not different (WT, n =10/6; Shank2 −/− , n =11/6). ( n ) Example traces of intrinsic Purkinje cell excitability as apparent from action potential firing evoked by 300 pA current injections. ( o ) No difference in evoked firing frequency relative to various levels of current injections (WT, n =10/5; Shank2 −/− , n =11/5, P =0.1, repeated-measures ANOVA). Inset barplot shows average slope of firing rate per current step ( P =1). Data are represented as mean±s.e.m. Two-sided t -tests were used, unless stated otherwise.
    Two Electrode Voltage Clamp Technique, supplied by dagan corp, used in various techniques. Bioz Stars score: 91/100, based on 78 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Robust LTD whole cell recording
    No changes in excitatory synaptic and intrinsic properties in Shank2 −/− Purkinje cells ex vivo . ( a ) <t>Recording</t> configuration for <t>voltage-clamp</t> recordings of PF–PC synaptic transmission. Inset: an example PF-EPSC. ( b – e ) With comparable holding current (at −65 mV) ( P =1) and input resistance (Ri) ( P =0.8), PC EPSC rise time ( P =0.2) and EPSC decay time ( P =0.3) are not different between WT ( n =9/6, cells per animals) and Shank2 −/− ( n =7/6). ( f ) Example EPSCs in response to 3, 6, 9, 12 and 15 μA stimulation. ( g , h ) Varying stimulation strength ( P =0.9, repeated-measures ANOVA) and inter-stimulus interval ( P =0.2, repeated-measures ANOVA) evoked comparable EPSC amplitude or facilitation (WT, n =11/3; Shank2 −/− 15/3). ( i ) Recording configuration for <t>whole-cell</t> recording. Inset: an example action potential. ( j – m ) Action potential threshold ( P =0.8), amplitude ( P =0.1), half-width ( P =0.7) and after-hyperpolarization ( P =0.2) were not different (WT, n =10/6; Shank2 −/− , n =11/6). ( n ) Example traces of intrinsic Purkinje cell excitability as apparent from action potential firing evoked by 300 pA current injections. ( o ) No difference in evoked firing frequency relative to various levels of current injections (WT, n =10/5; Shank2 −/− , n =11/5, P =0.1, repeated-measures ANOVA). Inset barplot shows average slope of firing rate per current step ( P =1). Data are represented as mean±s.e.m. Two-sided t -tests were used, unless stated otherwise.
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    3M Co optical patch clamp technique
    No changes in excitatory synaptic and intrinsic properties in Shank2 −/− Purkinje cells ex vivo . ( a ) <t>Recording</t> configuration for <t>voltage-clamp</t> recordings of PF–PC synaptic transmission. Inset: an example PF-EPSC. ( b – e ) With comparable holding current (at −65 mV) ( P =1) and input resistance (Ri) ( P =0.8), PC EPSC rise time ( P =0.2) and EPSC decay time ( P =0.3) are not different between WT ( n =9/6, cells per animals) and Shank2 −/− ( n =7/6). ( f ) Example EPSCs in response to 3, 6, 9, 12 and 15 μA stimulation. ( g , h ) Varying stimulation strength ( P =0.9, repeated-measures ANOVA) and inter-stimulus interval ( P =0.2, repeated-measures ANOVA) evoked comparable EPSC amplitude or facilitation (WT, n =11/3; Shank2 −/− 15/3). ( i ) Recording configuration for <t>whole-cell</t> recording. Inset: an example action potential. ( j – m ) Action potential threshold ( P =0.8), amplitude ( P =0.1), half-width ( P =0.7) and after-hyperpolarization ( P =0.2) were not different (WT, n =10/6; Shank2 −/− , n =11/6). ( n ) Example traces of intrinsic Purkinje cell excitability as apparent from action potential firing evoked by 300 pA current injections. ( o ) No difference in evoked firing frequency relative to various levels of current injections (WT, n =10/5; Shank2 −/− , n =11/5, P =0.1, repeated-measures ANOVA). Inset barplot shows average slope of firing rate per current step ( P =1). Data are represented as mean±s.e.m. Two-sided t -tests were used, unless stated otherwise.
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    Image Search Results


    Whole-cell voltage-clamp recordings of tardbp Y220X/Y220X + MO larvae displayed a higher frequency of mEPC events at 2 dpf. A, 60 second sample traces from 10 minute recordings from the following 4 treatment groups: WT, WT + MO, tardbp Y220X/Y220X and tardbp Y220X/Y220X + MO. B, Overlay of all mEPC events recorded during a 10 minute recording in each treatment group. C, Scatterplots graphing exponential decay constant ( tau ) vs amplitude distributions for 5 individual recordings from each treatment group. Plotted mEPCs can be divided into two populations; slow decay tau ( tau > 4 ms), activity detected from neighbouring muscle fibers, and fast decay tau ( tau

    Journal: PLoS ONE

    Article Title: Augmented quantal release of acetylcholine at the vertebrate neuromuscular junction following tdp-43 depletion

    doi: 10.1371/journal.pone.0177005

    Figure Lengend Snippet: Whole-cell voltage-clamp recordings of tardbp Y220X/Y220X + MO larvae displayed a higher frequency of mEPC events at 2 dpf. A, 60 second sample traces from 10 minute recordings from the following 4 treatment groups: WT, WT + MO, tardbp Y220X/Y220X and tardbp Y220X/Y220X + MO. B, Overlay of all mEPC events recorded during a 10 minute recording in each treatment group. C, Scatterplots graphing exponential decay constant ( tau ) vs amplitude distributions for 5 individual recordings from each treatment group. Plotted mEPCs can be divided into two populations; slow decay tau ( tau > 4 ms), activity detected from neighbouring muscle fibers, and fast decay tau ( tau

    Article Snippet: Whole-cell voltage-clamp recordings in fast-twitch muscle fibers As previously described [ ], zebrafish were anaesthetized in 0.02% tricaine (Sigma) dissolved in modified Evans solution containing the following (in mM): 134 NaCl, 2.9 KCl, 2.1 CaCl2 , 1.2 MgCl2 , 10 HEPES, and 10 glucose, adjusted to 290 mOsm, pH 7.8.

    Techniques: Mass Spectrometry, Activity Assay

    Whole‐cell targeting of Per1 ‐ EGFP +ve and EGFP ‐ve neurons in a living SCN slice. (A) Recording setup showing bright‐field image (4 ×) of a living coronal SCN slice (250 μm thick) taken at the mid‐rostro‐caudal level. (B) Image of Per1 ‐ EGFP +ve fluorescence signal (20 ×) taken from the slice shown in (A) with a FITC (488 nm, optimum for EGFP ) filter. Insets C1 and C2 are representative photomicrographs showing patch pipette targeting of EGFP −ve ( C1 , follow vertically) or Per1 ‐ EGFP +ve ( C2 ) neurons (40 ×) in the slice shown in A and B. Insets rows show bright‐field, fluorescence ( FITC ), and merged bright‐field and fluorescence images. (D and E) Spontaneous membrane excitability states of Per1 ‐ EGFP +ve (D) and EGFP −ve (E) SCN neurons over the day. (D) Example of typical current‐clamp traces from Per1 ‐ EGFP +ve SCN neurons recorded in the morning ( ZT 2–5) and afternoon ( ZT 6–10). Note that Per1 ‐ EGFP +ve neurons recorded in the morning are at moderate resting membrane potential ( RMP : ~ −45 mV ) and discharging action potentials ( AP s), while in the afternoon they become hyperexcited with RMP s of −25 to −35 mV and unable to generate AP s. These cells are either silent or generating depolarized low‐amplitude membrane oscillations ( DLAMO s). By contrast, EGFP −ve neurons (E) remain at moderate RMP throughout the day, firing AP s. Dashed lines in C indicate the outline of the patch pipette. SCN , suprachiasmatic nuclei; OX , optic chiasm; P , patch pipette. [Colour figure can be viewed at wileyonlinelibrary.com ].

    Journal: The European Journal of Neuroscience

    Article Title: Circadian regulation of mouse suprachiasmatic nuclei neuronal states shapes responses to orexin

    doi: 10.1111/ejn.13506

    Figure Lengend Snippet: Whole‐cell targeting of Per1 ‐ EGFP +ve and EGFP ‐ve neurons in a living SCN slice. (A) Recording setup showing bright‐field image (4 ×) of a living coronal SCN slice (250 μm thick) taken at the mid‐rostro‐caudal level. (B) Image of Per1 ‐ EGFP +ve fluorescence signal (20 ×) taken from the slice shown in (A) with a FITC (488 nm, optimum for EGFP ) filter. Insets C1 and C2 are representative photomicrographs showing patch pipette targeting of EGFP −ve ( C1 , follow vertically) or Per1 ‐ EGFP +ve ( C2 ) neurons (40 ×) in the slice shown in A and B. Insets rows show bright‐field, fluorescence ( FITC ), and merged bright‐field and fluorescence images. (D and E) Spontaneous membrane excitability states of Per1 ‐ EGFP +ve (D) and EGFP −ve (E) SCN neurons over the day. (D) Example of typical current‐clamp traces from Per1 ‐ EGFP +ve SCN neurons recorded in the morning ( ZT 2–5) and afternoon ( ZT 6–10). Note that Per1 ‐ EGFP +ve neurons recorded in the morning are at moderate resting membrane potential ( RMP : ~ −45 mV ) and discharging action potentials ( AP s), while in the afternoon they become hyperexcited with RMP s of −25 to −35 mV and unable to generate AP s. These cells are either silent or generating depolarized low‐amplitude membrane oscillations ( DLAMO s). By contrast, EGFP −ve neurons (E) remain at moderate RMP throughout the day, firing AP s. Dashed lines in C indicate the outline of the patch pipette. SCN , suprachiasmatic nuclei; OX , optic chiasm; P , patch pipette. [Colour figure can be viewed at wileyonlinelibrary.com ].

    Article Snippet: For whole‐cell patch‐clamp recordings, slices were transferred to a recording chamber mounted on the stage of an upright Olympus epi‐fluorescence microscope (BX51WI; Olympus, Japan), where they were continuously perfused (~ 3 mL/min) with recording aCSF.

    Techniques: Fluorescence, Transferring

    Anatomical and functional gap junction coupling in the developing mouse retina. A , Top , Projection of Neurobiotin-coupled neurons reconstructed from confocal sections taken from a P7 mouse retina. The ganglion cell in the center was filled with tracer; coupled ganglion cells (note the filled axons in the bottom left corner ) are observed in the GCL, and coupled amacrine cells are seen in both the GCL and INL. Scale bar, 20 μm. Bottom , Summary of Neurobiotin coupling throughout postnatal development before eye opening at P14; error bars are ±SD ( n = 47 filled RGCs). B , Top , Fluorescence image of the ganglion cell layer from a P2 mouse retina incubated in fluo-4 AM. Intracellular Ca 2+ elevations in neurons ( pseudocolored red ) are elicited by current steps (1 sec, 15 pA) injected into a recorded RGC ( circled in black ). The image is the average of responses to three repeated trials. Scale bar, 10 μm. Bottom left , The top traces are whole-cell current-clamp responses of an RGC to a current injection before and 10 min after bath application of 18α-GA to the retina. The bottom traces are examples of intracellular Ca 2+ fluorescence signals elicited in nonrecorded cells by these current injections (indicated by the arrow at point 4 ) observed before ( circles ) and after ( triangles ) 18α-GA treatment. Bottom right , Summary histogram plot of the percentage blockade of Δ F/F in 25 neurons in the GCL after current injection into nine RGCs; the x -axis represents the degree of block by 18α-GA, binned by 20% intervals, and the y -axis represents the percentage of cells for which each degree of blockade is observed.

    Journal: The Journal of Neuroscience

    Article Title: Potentiation of L-Type Calcium Channels Reveals Nonsynaptic Mechanisms that Correlate Spontaneous Activity in the Developing Mammalian Retina

    doi: 10.1523/JNEUROSCI.21-21-08514.2001

    Figure Lengend Snippet: Anatomical and functional gap junction coupling in the developing mouse retina. A , Top , Projection of Neurobiotin-coupled neurons reconstructed from confocal sections taken from a P7 mouse retina. The ganglion cell in the center was filled with tracer; coupled ganglion cells (note the filled axons in the bottom left corner ) are observed in the GCL, and coupled amacrine cells are seen in both the GCL and INL. Scale bar, 20 μm. Bottom , Summary of Neurobiotin coupling throughout postnatal development before eye opening at P14; error bars are ±SD ( n = 47 filled RGCs). B , Top , Fluorescence image of the ganglion cell layer from a P2 mouse retina incubated in fluo-4 AM. Intracellular Ca 2+ elevations in neurons ( pseudocolored red ) are elicited by current steps (1 sec, 15 pA) injected into a recorded RGC ( circled in black ). The image is the average of responses to three repeated trials. Scale bar, 10 μm. Bottom left , The top traces are whole-cell current-clamp responses of an RGC to a current injection before and 10 min after bath application of 18α-GA to the retina. The bottom traces are examples of intracellular Ca 2+ fluorescence signals elicited in nonrecorded cells by these current injections (indicated by the arrow at point 4 ) observed before ( circles ) and after ( triangles ) 18α-GA treatment. Bottom right , Summary histogram plot of the percentage blockade of Δ F/F in 25 neurons in the GCL after current injection into nine RGCs; the x -axis represents the degree of block by 18α-GA, binned by 20% intervals, and the y -axis represents the percentage of cells for which each degree of blockade is observed.

    Article Snippet: Whole-cell current-clamp recordings were made from visualized ganglion cells (40× water-immersion objective;Olympus Optical).

    Techniques: Functional Assay, Fluorescence, Incubation, Size-exclusion Chromatography, Injection, Blocking Assay

    Effects of μ-TRTX-Ca2a on Na v 1.2–Na v 1.9 channels. (A–H) Representative Na v 1.2–Na v 1.9 current traces before (black) and after (red) addition of Ca2a. Ca2a at 1 μM inhibited Na v 1.2–Na v 1.3, Na v 1.6, and Na v 1.7. 10 μM Ca2a showed no obvious effect on Na v 1.4–Na v 1.5 or Na v 1.8–Na v 1.9 current. Inset above panel (A) shows the pulse protocol for recording Na v 1.2–Na v 1.7 channel currents. Inset above panel (G) shows the pulse protocol for recording Na v 1.8 channel current. Inset above panel (H) shows the pulse protocol for recording Na v 1.9 channel current. (I) Concentration-response curves of Ca2a at Na v 1.2–Na v 1.3, Na v 1.6, and Na v 1.7 assessed by whole-cell patch-clamp experiments. Data are mean ± SEM, with n = 4–7 cells per data point.

    Journal: Frontiers in Pharmacology

    Article Title: Discovery of a Novel Nav1.7 Inhibitor From Cyriopagopus albostriatus Venom With Potent Analgesic Efficacy

    doi: 10.3389/fphar.2018.01158

    Figure Lengend Snippet: Effects of μ-TRTX-Ca2a on Na v 1.2–Na v 1.9 channels. (A–H) Representative Na v 1.2–Na v 1.9 current traces before (black) and after (red) addition of Ca2a. Ca2a at 1 μM inhibited Na v 1.2–Na v 1.3, Na v 1.6, and Na v 1.7. 10 μM Ca2a showed no obvious effect on Na v 1.4–Na v 1.5 or Na v 1.8–Na v 1.9 current. Inset above panel (A) shows the pulse protocol for recording Na v 1.2–Na v 1.7 channel currents. Inset above panel (G) shows the pulse protocol for recording Na v 1.8 channel current. Inset above panel (H) shows the pulse protocol for recording Na v 1.9 channel current. (I) Concentration-response curves of Ca2a at Na v 1.2–Na v 1.3, Na v 1.6, and Na v 1.7 assessed by whole-cell patch-clamp experiments. Data are mean ± SEM, with n = 4–7 cells per data point.

    Article Snippet: Whole-cell patch-clamp recordings were performed at room temperature (20–25°C) using an EPC 10 USB Patch Clamp Amplifier (HEKA, Elektronik, Lambrecht, Germany).

    Techniques: Concentration Assay, Patch Clamp

    Ca 2+ dependence of anion selectivity for TMEM16A is abolished by mutations of basic residues. ( A ) Currents recorded from voltage ramps from −80 to +80 mV in WT TMEM16A in whole-cell patch clamp with 400 nM Ca 2+ in the pipette. Intracellular solution contained 140 mM NaCl, whereas extracellular ion concentrations are as indicated. ( B ) Currents recorded from voltage ramps from −80 to +80 mV in WT TMEM16A with 1 mMCa 2+ in the pipette. ( C ) Permeability ratios are determined by solving the Goldman–Hodgkin–Katz voltage equation for each solution compared with chloride in the indicated concentrations of Ca 2+ . Permeability ratios for thiocyanate over chloride are shown for alanine mutants at ( D ) 400 nM Ca 2+ and ( E ) 1 mM Ca 2+ . N.S., not significant. *** P

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Four basic residues critical for the ion selectivity and pore blocker sensitivity of TMEM16A calcium-activated chloride channels

    doi: 10.1073/pnas.1502291112

    Figure Lengend Snippet: Ca 2+ dependence of anion selectivity for TMEM16A is abolished by mutations of basic residues. ( A ) Currents recorded from voltage ramps from −80 to +80 mV in WT TMEM16A in whole-cell patch clamp with 400 nM Ca 2+ in the pipette. Intracellular solution contained 140 mM NaCl, whereas extracellular ion concentrations are as indicated. ( B ) Currents recorded from voltage ramps from −80 to +80 mV in WT TMEM16A with 1 mMCa 2+ in the pipette. ( C ) Permeability ratios are determined by solving the Goldman–Hodgkin–Katz voltage equation for each solution compared with chloride in the indicated concentrations of Ca 2+ . Permeability ratios for thiocyanate over chloride are shown for alanine mutants at ( D ) 400 nM Ca 2+ and ( E ) 1 mM Ca 2+ . N.S., not significant. *** P

    Article Snippet: Whole-cell patch-clamp recordings were performed using borosilicate capillary glass electrodes (Sutter Instrument) polished to a tip resistance of 3–5 MΩ.

    Techniques: Patch Clamp, Transferring, Permeability

    Alanine scan of putative pore-lining basic residues identifies five that significantly increase permeability ratios in bi-ionic condition. ( A ) Proposed topology of TMEM16A channel with basic residues within the putative pore region or previously implicated in permeation indicated at their predicted locations. ( B ) Representative currents recorded from voltage ramps from −80 to +80 mV in whole-cell patch clamp in the constructs indicated. Color coding indicates extracellular ion species (all at 140 mM). Black, NaCl; blue, NaI; green, NaSCN; red, NaBr. Permeability ratios for all alanine mutants as determined by solving the Goldman–Hodgkin–Katz voltage equation for ( C ) bromide, ( D ) iodide, and ( E ) thiocyanate compared with chloride for each construct. Ratios are compared using one-way ANOVA followed by Tukey’s posthoc test. Statistical comparison of WT TMEM16A in whole-cell mode with other constructs is indicated with the top bar. ***Significant difference ( P

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Four basic residues critical for the ion selectivity and pore blocker sensitivity of TMEM16A calcium-activated chloride channels

    doi: 10.1073/pnas.1502291112

    Figure Lengend Snippet: Alanine scan of putative pore-lining basic residues identifies five that significantly increase permeability ratios in bi-ionic condition. ( A ) Proposed topology of TMEM16A channel with basic residues within the putative pore region or previously implicated in permeation indicated at their predicted locations. ( B ) Representative currents recorded from voltage ramps from −80 to +80 mV in whole-cell patch clamp in the constructs indicated. Color coding indicates extracellular ion species (all at 140 mM). Black, NaCl; blue, NaI; green, NaSCN; red, NaBr. Permeability ratios for all alanine mutants as determined by solving the Goldman–Hodgkin–Katz voltage equation for ( C ) bromide, ( D ) iodide, and ( E ) thiocyanate compared with chloride for each construct. Ratios are compared using one-way ANOVA followed by Tukey’s posthoc test. Statistical comparison of WT TMEM16A in whole-cell mode with other constructs is indicated with the top bar. ***Significant difference ( P

    Article Snippet: Whole-cell patch-clamp recordings were performed using borosilicate capillary glass electrodes (Sutter Instrument) polished to a tip resistance of 3–5 MΩ.

    Techniques: Permeability, Patch Clamp, Construct

    Alanine mutants that alter anion selectivity also affect potency of pore blockers. ( A ) Representative currents recorded from voltage ramps from −80 to +80 mV in whole-cell patch clamp for TMEM16A in NaCl bath solution with increasing concentrations of blocker NTTP as indicated. ( B ) Concentration–response curves are plotted for NTTP for TMEM16A and alanine mutants that alter anion selectivity. ( C ) Representative currents recorded from voltage ramps from −80 to +80 mV in whole-cell patch clamp for TMEM16A in NaCl bath solution with increasing concentrations of blocker 1PBC as indicated. ( D ) Concentration–response curves are plotted for 1PBC for TMEM16A and alanine mutants that alter anion selectivity.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Four basic residues critical for the ion selectivity and pore blocker sensitivity of TMEM16A calcium-activated chloride channels

    doi: 10.1073/pnas.1502291112

    Figure Lengend Snippet: Alanine mutants that alter anion selectivity also affect potency of pore blockers. ( A ) Representative currents recorded from voltage ramps from −80 to +80 mV in whole-cell patch clamp for TMEM16A in NaCl bath solution with increasing concentrations of blocker NTTP as indicated. ( B ) Concentration–response curves are plotted for NTTP for TMEM16A and alanine mutants that alter anion selectivity. ( C ) Representative currents recorded from voltage ramps from −80 to +80 mV in whole-cell patch clamp for TMEM16A in NaCl bath solution with increasing concentrations of blocker 1PBC as indicated. ( D ) Concentration–response curves are plotted for 1PBC for TMEM16A and alanine mutants that alter anion selectivity.

    Article Snippet: Whole-cell patch-clamp recordings were performed using borosilicate capillary glass electrodes (Sutter Instrument) polished to a tip resistance of 3–5 MΩ.

    Techniques: Patch Clamp, Concentration Assay

    Characterization of two pore blockers from a high-throughput small molecule screen. ( A ) Structure of TMEM16A inhibitor (PubChem SID 50085892) NTTP. ( B ) Representative currents recorded from voltage ramps from −80 to +80 mV in whole-cell patch clamp for TMEM16A in NaCl bath solution (black trace) and bath solution with 30 μM NTTP (red trace). ( C ) Voltage dependence of block is calculated at positive potentials from the Woodhull equation, and effective block distance from the external face is shown as zδ . ( D ) Sample traces showing blocker onset and washout in ( Upper ) 140 mM NaCl solution and ( Lower ) low ionic solution containing 14 mM NaCl supplemented with sucrose. ( E ) Structure of TMEM16A inhibitor (PubChem SID 49642647) 1PBC. ( F ) Representative currents recorded from voltage ramps from −80 to +80 mV in whole-cell patch clamp for TMEM16A in NaCl bath solution (black trace) and bath solution with 30 μM 1PBC (blue trace). ( G ) Voltage dependence of block is calculated at positive potentials from the Woodhull equation. ( H ) Sample traces showing blocker onset and washout when membrane potential is held at +60 mV in ( Upper ) 140 mM NaCl solution and ( Lower ) low ionic solution containing 14 mM NaCl supplemented with sucrose. ( I ) Time constants fit to traces in D for block and unblock on washout of NTTP. ( J ) Time constants from double exponentials fit to blocking time course for traces in I for 1PBC with either 140 or 14 mM NaCl. ( K ) Time constants from double exponentials fit to unblock time course for traces in I . K , Inset shows expanded view of comparison of fast time constants. *** P

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Four basic residues critical for the ion selectivity and pore blocker sensitivity of TMEM16A calcium-activated chloride channels

    doi: 10.1073/pnas.1502291112

    Figure Lengend Snippet: Characterization of two pore blockers from a high-throughput small molecule screen. ( A ) Structure of TMEM16A inhibitor (PubChem SID 50085892) NTTP. ( B ) Representative currents recorded from voltage ramps from −80 to +80 mV in whole-cell patch clamp for TMEM16A in NaCl bath solution (black trace) and bath solution with 30 μM NTTP (red trace). ( C ) Voltage dependence of block is calculated at positive potentials from the Woodhull equation, and effective block distance from the external face is shown as zδ . ( D ) Sample traces showing blocker onset and washout in ( Upper ) 140 mM NaCl solution and ( Lower ) low ionic solution containing 14 mM NaCl supplemented with sucrose. ( E ) Structure of TMEM16A inhibitor (PubChem SID 49642647) 1PBC. ( F ) Representative currents recorded from voltage ramps from −80 to +80 mV in whole-cell patch clamp for TMEM16A in NaCl bath solution (black trace) and bath solution with 30 μM 1PBC (blue trace). ( G ) Voltage dependence of block is calculated at positive potentials from the Woodhull equation. ( H ) Sample traces showing blocker onset and washout when membrane potential is held at +60 mV in ( Upper ) 140 mM NaCl solution and ( Lower ) low ionic solution containing 14 mM NaCl supplemented with sucrose. ( I ) Time constants fit to traces in D for block and unblock on washout of NTTP. ( J ) Time constants from double exponentials fit to blocking time course for traces in I for 1PBC with either 140 or 14 mM NaCl. ( K ) Time constants from double exponentials fit to unblock time course for traces in I . K , Inset shows expanded view of comparison of fast time constants. *** P

    Article Snippet: Whole-cell patch-clamp recordings were performed using borosilicate capillary glass electrodes (Sutter Instrument) polished to a tip resistance of 3–5 MΩ.

    Techniques: High Throughput Screening Assay, Patch Clamp, Blocking Assay

    A comparison of normalized peak current amplitudes for wild-type and mutant hNa v 1.5 channels expressed by two different expression systems. Whole-cell currents were recorded by the patch clamp technique (HEK293 cells) and by the two-microelectrode voltage clamp technique (oocytes) as described in “ Methods ”. L212P and T220I exhibited similar relative expression levels in both systems. With E161K, D1275N, and P1298L, whole-cell currents, normalized to hNa v 1.5 values, were significantly smaller for the oocyte system (* indicates p

    Journal: PLoS ONE

    Article Title: Multiple Loss-of-Function Mechanisms Contribute to SCN5A-Related Familial Sick Sinus Syndrome

    doi: 10.1371/journal.pone.0010985

    Figure Lengend Snippet: A comparison of normalized peak current amplitudes for wild-type and mutant hNa v 1.5 channels expressed by two different expression systems. Whole-cell currents were recorded by the patch clamp technique (HEK293 cells) and by the two-microelectrode voltage clamp technique (oocytes) as described in “ Methods ”. L212P and T220I exhibited similar relative expression levels in both systems. With E161K, D1275N, and P1298L, whole-cell currents, normalized to hNa v 1.5 values, were significantly smaller for the oocyte system (* indicates p

    Article Snippet: Whole-oocyte Na+ currents were recorded with the two-microelectrode voltage clamp technique using an OC725C amplifier (Warner Instruments, Hamden, CT, USA), as previously described .

    Techniques: Mutagenesis, Expressing, Patch Clamp

    Insulin induced a slight hyperpolarization in DMV neurons and significantly decreased whole cell input resistance in neurons that hyperpolarized. A : sample traces of current-clamp recordings from the same unidentified DMV neuron in aCSF + TTX (2 μM)

    Journal: American Journal of Physiology - Regulatory, Integrative and Comparative Physiology

    Article Title: Insulin reduces excitation in gastric-related neurons of the dorsal motor nucleus of the vagus

    doi: 10.1152/ajpregu.00276.2012

    Figure Lengend Snippet: Insulin induced a slight hyperpolarization in DMV neurons and significantly decreased whole cell input resistance in neurons that hyperpolarized. A : sample traces of current-clamp recordings from the same unidentified DMV neuron in aCSF + TTX (2 μM)

    Article Snippet: After an equilibration period of ∼1 h, whole cell voltage-clamp recordings were obtained from DMV neurons under visual guidance on an upright, fixed-stage microscope equipped with infrared illumination and differential interference contrast (IR-DIC) and epifluorescence optics (BX51WI, Olympus).

    Techniques:

    Ex vivo — in vitro whole-cell patch recordings of Cy3- Wisteria floribunda agglutinin (WFA)-labeled fast-spiking interneurons (FSIs) after transcranial magnetic stimulation (TMS). (A) Drawing showing placement and orientation of the figure-of-eight coil used to apply the intermittent theta-burst (iTBS) protocol (see B ) to the rat brain. At the orientation shown, a mediolaterally oriented electric field is induced within the brain, optimally suited to depolarize axons of the corpus callosum. (C) Red fluorescent Cy3-WFA-labeled perineuronal net of a cortical FSIs revealed by fluorescence microscopy. (D) The same neuron shown in infrared phase contrast illumination. (E) Same as in (D) but with the patch pipette attached to the cell. (F) Cell filled with Alexa488.

    Journal: Frontiers in Neural Circuits

    Article Title: Intermittent Theta-Burst Transcranial Magnetic Stimulation Alters Electrical Properties of Fast-Spiking Neocortical Interneurons in an Age-Dependent Fashion

    doi: 10.3389/fncir.2016.00022

    Figure Lengend Snippet: Ex vivo — in vitro whole-cell patch recordings of Cy3- Wisteria floribunda agglutinin (WFA)-labeled fast-spiking interneurons (FSIs) after transcranial magnetic stimulation (TMS). (A) Drawing showing placement and orientation of the figure-of-eight coil used to apply the intermittent theta-burst (iTBS) protocol (see B ) to the rat brain. At the orientation shown, a mediolaterally oriented electric field is induced within the brain, optimally suited to depolarize axons of the corpus callosum. (C) Red fluorescent Cy3-WFA-labeled perineuronal net of a cortical FSIs revealed by fluorescence microscopy. (D) The same neuron shown in infrared phase contrast illumination. (E) Same as in (D) but with the patch pipette attached to the cell. (F) Cell filled with Alexa488.

    Article Snippet: Single cell responses obtained with whole-cell patch clamp recordings were amplified using a PC-501A amplifier (Warner Instruments, Hamden, CT, USA), recorded and digitized at a sampling frequency of 2.73 kHz using WinWCP Software (version 4.6.7, Strathclyde University, Scotland) and a Digidata 1440A interface (Axon CNS, Molecular Devices, Sunnyvale, CA, USA) before being finally stored on a hard disk for off-line analysis.

    Techniques: Ex Vivo, In Vitro, Labeling, Fluorescence, Microscopy, Transferring

    Cellular pH and Chloride Transport Studies (A) ClC-7 p.Tyr715Cys expression causes increased current in Xenopus oocytes. Two-electrode voltage-clamp current traces are shown for uninjected (black), WT ClC-7 PM (blue), and p.Tyr715Cys ClC-7 PM (red) in response to a series of 1 s voltage steps from −100 mV to + 80 mV in increments of 20 mV, followed by a step to −80 mV for 1.4 s before return to a holding potential of −30 mV. (B) Bar graphs of current (+/− SEM, measured at +80 mV) or surface expression of uninjected (black), WT ClC-7 PM , or p.Tyr715Cys ClC-7 PM . For current measurements, n = 3 (uninjected), 8 (WT), or 6 (p.Tyr715Cys) from a single batch of oocytes; the experiment was reproduced 11 times in separate oocyte batches with essentially identical results. For surface expression, five separate surface biotinylation experiments were performed (with 20 each of WT and p.Tyr715Cys oocytes); intensities were normalized by the WT band intensity and averaged. The currents shown in (A) were from one of the same batches of oocytes used for the surface-expression experiments. (C) Lysosomal pH in control and proband fibroblasts was measured with OG488 ratiometric imaging. Raw fluorescence ratios are shown for individual cells (pale circles) and averages (+/− SEM) of n = ∼50 cells (dark circles) from two independent experiments each on control neonatal fibroblasts (1.54 ± 0.1) or primary fibroblasts from either proband (proband 1, 1.30 ± 0.07; proband 2, 1.28 ± 0.08). Ratios from bafilomycin-treated cells from each are also shown as a negative control. The lower the 490/440nm fluorescence ratio, the lower the pH. An image of an OG488-treated proband cell is shown in the inset. ). Error bars indicate SEM; p values as shown. (E) Cultured fibroblasts incubated with LysoTracker Red DND-99. Fluorescent intensity was significantly greater in cells of probands 1 and 2 than in control cells. Nuclei are stained with Hoechst (blue channel). (F) Mean (+/− SEM) integrated intensity of LysoTracker Red DND-99 (red channel) per cell is plotted in relative fluorescence units (RFU); values are 10 7 . Nuclei are stained with Hoechst (blue channel). Fluorescent intensity was significantly greater in cells of probands 1 and 2 than in control cells. Abbreviations are as follows: SEM, standard error the mean; WT, wild-type.

    Journal: American Journal of Human Genetics

    Article Title: Lysosomal Storage and Albinism Due to Effects of a De Novo CLCN7 Variant on Lysosomal Acidification

    doi: 10.1016/j.ajhg.2019.04.008

    Figure Lengend Snippet: Cellular pH and Chloride Transport Studies (A) ClC-7 p.Tyr715Cys expression causes increased current in Xenopus oocytes. Two-electrode voltage-clamp current traces are shown for uninjected (black), WT ClC-7 PM (blue), and p.Tyr715Cys ClC-7 PM (red) in response to a series of 1 s voltage steps from −100 mV to + 80 mV in increments of 20 mV, followed by a step to −80 mV for 1.4 s before return to a holding potential of −30 mV. (B) Bar graphs of current (+/− SEM, measured at +80 mV) or surface expression of uninjected (black), WT ClC-7 PM , or p.Tyr715Cys ClC-7 PM . For current measurements, n = 3 (uninjected), 8 (WT), or 6 (p.Tyr715Cys) from a single batch of oocytes; the experiment was reproduced 11 times in separate oocyte batches with essentially identical results. For surface expression, five separate surface biotinylation experiments were performed (with 20 each of WT and p.Tyr715Cys oocytes); intensities were normalized by the WT band intensity and averaged. The currents shown in (A) were from one of the same batches of oocytes used for the surface-expression experiments. (C) Lysosomal pH in control and proband fibroblasts was measured with OG488 ratiometric imaging. Raw fluorescence ratios are shown for individual cells (pale circles) and averages (+/− SEM) of n = ∼50 cells (dark circles) from two independent experiments each on control neonatal fibroblasts (1.54 ± 0.1) or primary fibroblasts from either proband (proband 1, 1.30 ± 0.07; proband 2, 1.28 ± 0.08). Ratios from bafilomycin-treated cells from each are also shown as a negative control. The lower the 490/440nm fluorescence ratio, the lower the pH. An image of an OG488-treated proband cell is shown in the inset. ). Error bars indicate SEM; p values as shown. (E) Cultured fibroblasts incubated with LysoTracker Red DND-99. Fluorescent intensity was significantly greater in cells of probands 1 and 2 than in control cells. Nuclei are stained with Hoechst (blue channel). (F) Mean (+/− SEM) integrated intensity of LysoTracker Red DND-99 (red channel) per cell is plotted in relative fluorescence units (RFU); values are 10 7 . Nuclei are stained with Hoechst (blue channel). Fluorescent intensity was significantly greater in cells of probands 1 and 2 than in control cells. Abbreviations are as follows: SEM, standard error the mean; WT, wild-type.

    Article Snippet: After incubating the oocytes for 5 days at 17°C, we measured currents through the use of standard two-electrode voltage clamp techniques (OC-725C; Warner Instruments) at room temperature.

    Techniques: Expressing, Imaging, Fluorescence, Negative Control, Cell Culture, Incubation, Staining

    No changes in excitatory synaptic and intrinsic properties in Shank2 −/− Purkinje cells ex vivo . ( a ) Recording configuration for voltage-clamp recordings of PF–PC synaptic transmission. Inset: an example PF-EPSC. ( b – e ) With comparable holding current (at −65 mV) ( P =1) and input resistance (Ri) ( P =0.8), PC EPSC rise time ( P =0.2) and EPSC decay time ( P =0.3) are not different between WT ( n =9/6, cells per animals) and Shank2 −/− ( n =7/6). ( f ) Example EPSCs in response to 3, 6, 9, 12 and 15 μA stimulation. ( g , h ) Varying stimulation strength ( P =0.9, repeated-measures ANOVA) and inter-stimulus interval ( P =0.2, repeated-measures ANOVA) evoked comparable EPSC amplitude or facilitation (WT, n =11/3; Shank2 −/− 15/3). ( i ) Recording configuration for whole-cell recording. Inset: an example action potential. ( j – m ) Action potential threshold ( P =0.8), amplitude ( P =0.1), half-width ( P =0.7) and after-hyperpolarization ( P =0.2) were not different (WT, n =10/6; Shank2 −/− , n =11/6). ( n ) Example traces of intrinsic Purkinje cell excitability as apparent from action potential firing evoked by 300 pA current injections. ( o ) No difference in evoked firing frequency relative to various levels of current injections (WT, n =10/5; Shank2 −/− , n =11/5, P =0.1, repeated-measures ANOVA). Inset barplot shows average slope of firing rate per current step ( P =1). Data are represented as mean±s.e.m. Two-sided t -tests were used, unless stated otherwise.

    Journal: Nature Communications

    Article Title: Dysfunctional cerebellar Purkinje cells contribute to autism-like behaviour in Shank2-deficient mice

    doi: 10.1038/ncomms12627

    Figure Lengend Snippet: No changes in excitatory synaptic and intrinsic properties in Shank2 −/− Purkinje cells ex vivo . ( a ) Recording configuration for voltage-clamp recordings of PF–PC synaptic transmission. Inset: an example PF-EPSC. ( b – e ) With comparable holding current (at −65 mV) ( P =1) and input resistance (Ri) ( P =0.8), PC EPSC rise time ( P =0.2) and EPSC decay time ( P =0.3) are not different between WT ( n =9/6, cells per animals) and Shank2 −/− ( n =7/6). ( f ) Example EPSCs in response to 3, 6, 9, 12 and 15 μA stimulation. ( g , h ) Varying stimulation strength ( P =0.9, repeated-measures ANOVA) and inter-stimulus interval ( P =0.2, repeated-measures ANOVA) evoked comparable EPSC amplitude or facilitation (WT, n =11/3; Shank2 −/− 15/3). ( i ) Recording configuration for whole-cell recording. Inset: an example action potential. ( j – m ) Action potential threshold ( P =0.8), amplitude ( P =0.1), half-width ( P =0.7) and after-hyperpolarization ( P =0.2) were not different (WT, n =10/6; Shank2 −/− , n =11/6). ( n ) Example traces of intrinsic Purkinje cell excitability as apparent from action potential firing evoked by 300 pA current injections. ( o ) No difference in evoked firing frequency relative to various levels of current injections (WT, n =10/5; Shank2 −/− , n =11/5, P =0.1, repeated-measures ANOVA). Inset barplot shows average slope of firing rate per current step ( P =1). Data are represented as mean±s.e.m. Two-sided t -tests were used, unless stated otherwise.

    Article Snippet: Whole-cell patch clamp recording were performed with an EPC9 amplifier (HEKA Electronics, Lambrecht, Germany).

    Techniques: Ex Vivo, Transmission Assay