Journal: PLoS ONE

Article Title: Action Potential Modulation in CA1 Pyramidal Neuron Axons Facilitates OLM Interneuron Activation in Recurrent Inhibitory Microcircuits of Rat Hippocampus

doi: 10.1371/journal.pone.0113124

Figure Lengend Snippet: (A) Top, K + current evoked in outside-out patches from the axon (434 µm) of CA1 pyramidal neuron by 200-ms pulses to 50 mV in the absence (Black trace, I Control ) and presence (Gray trace, I 100 nM α-DTX ) of 100 nM α-DTX. Middle, the blue trace represents the α-DTX-sensitive current component obtained by digital subtraction. Bottom, bar plot of the average time constant of inactivation (n = 10). (B) Summary bar graph showing the effect of 100 nM α-DTX in axonal patches (left, P<0.001, n = 13) and somatic patches (right, P>0.1, n = 6). Bars indicate mean ± SEM; circles denote individual experiments. Data points for the same experimental conditions are connected by lines. (C) Recovery of α-DTX-sensitive K + channel from inactivation. Stimulation protocol and example traces of recovery from inactivation, as probed with a double-pulse protocol (100-ms prepulse to –120 mV, 150-ms conditioning pulses and 30-ms test pulses to +50 mV, separated by a recovery interval of variable duration at –120 mV). The holding potential before and after the pulse protocol was –90 mV. Axonal patch is 388 µm from the soma. (D) Summary plot of the amplitude of the peak current evoked by the test pulse, divided by that evoked by the conditioning pulse, plotted against interpulse interval. Data points represent means from 5 patches. Red curves represent double-exponential fit to the data points. (E, F) Gating properties of α-DTX-sensitive axonal K + channels. Stimulation protocol and traces of activation and steady-state inactivation of α-DTX-sensitive K + channels in CA1 pyramidal neuron axons. (E) To probe steady-state activation, a 100-ms prepulse to –120 mV was followed by a 200-ms test pulse to various potentials (–120 to 50 mV). (F) To test steady-state inactivation, a 5-s prepulse to various potentials (–120 to 0 mV) was followed by a 200-ms test pulse to 50 mV. Axonal patch is 423 µm from the soma. (G) Activation (blue circles) and steady-state inactivation (black squares) curves. Conductance values were normalized to the maximal value. Data points represent means from 8 patches for activation curve and 6 patches for steady-state inactivation. Blue curves represent Boltzmann functions fit to the data points. For the activation curve (blue line), the midpoint potential was –12 mV and the sloe factor 26.4 mV. For the inactivation curve (black line), the midpoint potential was –64 mV and the slope factor 15.2 mV. (H) Length constant of the CA1 pyramidal neuron axons. Plot of axonal to somatic voltage deflection during hyperpolarizing current pulses (1 s, –30 pA) applied at the soma, plotted against distance. Each data point represents a simultaneous axon–soma recording. Red line represents a fit of data points with an exponential function, resulting in a mean length constant of 712 µm. Note that the long length constant results in particularly efficient propagation of subthreshold membrane potential changes from the soma to the axon. (Inset) voltage changes recorded in the soma (black) and the axon (blue) during hyperpolarizing current pulses applied at the soma. Axonal recording site is 705 µm from the soma. Traces shown represent average of 20 (A, black trace), 22 (A, gray trace), 2 (C), 3 (E), and 2 (F) single sweeps. Transient inward Na + currents at the beginning of the pulse are truncated. Error bars, SEM.

Article Snippet: For paired recordings, K-gluconate and EGTA concentrations were modified to 135 mM and 0.1 mM, respectively. α-Dendrotoxin (α-DTX) was purchased from Alomone labs. For experiments with α-DTX, 0.1% bovine serum albumin (BSA) was added to the extracellular solutions to prevent peptide adsorption.

Techniques: Activation Assay

Journal: PLoS ONE

Article Title: Action Potential Modulation in CA1 Pyramidal Neuron Axons Facilitates OLM Interneuron Activation in Recurrent Inhibitory Microcircuits of Rat Hippocampus

doi: 10.1371/journal.pone.0113124

Figure Lengend Snippet: (A) Block of Kv1 channels enhances transmission. Top, unitary EPSCs at CA1 PN–O-LM IN synapses evoked by single presynaptic APs in control conditions and in the presence of 100 nM α-DTX. Traces represent averages from 10 single sweeps. Bottom, summary bar graph of the effects of α-DTX on EPSC peak amplitude. Note that α-DTX markedly increased synaptic efficacy. (B) Block of Kv1 channels reduces facilitation of transmission. Top, EPSCs at CA1 PN–O-LM IN synapses evoked by trains of five presynaptic APs in control conditions (left) and in the presence of 100 nM α-DTX (right). Bottom, facilitation ratio (EPSC n /EPSC 1 ), plotted against stimulus number, in control conditions (squares) and in the presence of α-DTX (circles). Somatic holding potential –60 mV. (C) Block of Kv1 channels abolishes static analog modulation of transmission. Top, unitary EPSCs at CA1 PN–O-LM IN synapses evoked by single presynaptic APs in control conditions and in the presence of 100 nM α-DTX. Presynaptic membrane potential were held at –60 mV (left; same recording as in (A)), and –50 mV (right). Bottom, summary bar graph of the effects of α-DTX on EPSC peak amplitude (EPSC DTX /EPSC Control ) for two different presynaptic holding potential (black, –60 mV; red, –50 mV). Note that α-DTX occluded the effects of changing membrane potential in the presynaptic neuron. (D) Block of Kv1 channels broadens the axonal AP. Top, axonal AP traces in control conditions and in the presence of 100 nM α-DTX. Bottom, summary bar graph showing the effects of α-DTX on half-duration of somatic and axonal AP. Axonal recording site is 264 µm from the soma. Note that α-DTX selectively increased axonal AP duration. (E) Block of Kv1 channels reduces activity-dependent AP broadening. Top, superposition of 1 st , 5th, and 50 th axonal AP in control conditions (left) and in the presence of 100 nM α-DTX (right). Bottom, plot of axonal AP half-width against stimulus number in control conditions (squares) and in the presence of 100 nM α-DTX (circles). Somatic holding potential –60 mV. Data from 7 recordings at distances of 200 to 500 µm. Axonal recording site is 264 µm from the soma. (F) Block of Kv1 channels reduces static AP broadening. Top, superposition of axonal APs in control conditions and in the presence of 100 nM α-DTX at –60 mV (left; same recording as in (D)), and –50 mV (right). Bottom, summary bar graph of the effects of α-DTX on AP broadening at –60 mV (black) and –50 mV (red). Note that the depolarization-induced AP broadening reduced the effect of α-DTX. Axonal recording site is 264 µm from the soma. Bars indicate mean ± SEM. Open circles represent data from individual experiments. Data from the same experiment or for the same experimental conditions were connected by lines. *0.01≤P<0.05.

Article Snippet: For paired recordings, K-gluconate and EGTA concentrations were modified to 135 mM and 0.1 mM, respectively. α-Dendrotoxin (α-DTX) was purchased from Alomone labs. For experiments with α-DTX, 0.1% bovine serum albumin (BSA) was added to the extracellular solutions to prevent peptide adsorption.

Techniques: Blocking Assay, Transmission Assay, Activity Assay

Journal: Cell Reports

Article Title: Large, Stable Spikes Exhibit Differential Broadening in Excitatory and Inhibitory Neocortical Boutons

doi: 10.1016/j.celrep.2020.108612

Figure Lengend Snippet: Presynaptic Spike Amplitude Is Independent of Potassium Channels and Stable during Synaptic Scaling (A) Left: example action potential recorded from cultured boutons with 100 nM DTX-K included in the bath perfusion. Right: example action potential recorded from cultured boutons with 100 nM DTX-K + 1 mM TEA included in the bath perfusion. Representative control action potential recorded with quartz pipettes at physiological temperature (black, same as in Figure 5 B) is provided for comparison. Traces were aligned to the point of steepest rise during depolarization. (B) Left: peak potential of action potentials recorded under the conditions illustrated in (A). Right: half-duration of action potentials recorded under the conditions illustrated in (A) (color code as in A; bar graphs as mean ± SEM; n indicates number of recordings from individual boutons). (C) Example action potentials recorded from cultured boutons under control conditions (same as in Figure 5 B ) and after 48 h exposure to 1 μM TTX or 100 μM PTX (from left to right, respectively). (D) Left: overshoot of action potentials recorded under the conditions illustrated in (C). Right: half-duration of action potentials recorded under the conditions illustrated in (C) (color code as in C; bar graphs as mean ± SEM; n indicates number of recordings from individual boutons; control data same as in Figure 5 E). See also Figure S5 .

Article Snippet: DTX-K , Alomone Labs , Cat# D-400.

Techniques: Cell Culture

Journal: Cell Reports

Article Title: Large, Stable Spikes Exhibit Differential Broadening in Excitatory and Inhibitory Neocortical Boutons

doi: 10.1016/j.celrep.2020.108612

Figure Lengend Snippet:

Article Snippet: DTX-K , Alomone Labs , Cat# D-400.

Techniques: Recombinant, Software

Journal: Scientific Reports

Article Title: Kv1 potassium channels control action potential firing of putative GABAergic deep cerebellar nuclear neurons

doi: 10.1038/s41598-020-63583-7

Figure Lengend Snippet: The spontaneous and evoked changes in AP firing activity of GAD + DCNs are sensitive to DTX-alpha. ( A ) From left to right: The traces illustrate typical example of spontaneous action potentials recorded from one GAD + DCN under control conditions (red), DTX-alpha (100 nM, DTX-alpha, blue), and with current injection to match the control ISI-Vm during DTX-alpha application (DTX-alpha& DC, green see text for details). (For comparison, same neuron as in Fig. ). The black circles on top of each spike represent the instantaneous frequency (IF, Hz). Note the scale on the left indicate both the membrane potential (Vm) and the IF. A’ detail of A in expanded time scale. The color code used here applies to all figures. ( B ) Bar plots of the mean spontaneous frequency under control and during DTX-alpha application and changes in individual results indicated by the gray lines (see details in main text). ( C ) Top, left: typical examples from the same GAD + DCNs illustrating changes in spiking activity induced by injection of depolarizing current pulses of 1 second duration and increasing current intensity (bottom to top), used to determine the maximum repetitive frequency (MRF), defined as the maximum frequency at steady state before depolarizing block (middle trace, detail shown in the inset) (see methods for further details). ( D ) Mean MRF for GAD + DCNs before (control) and during DTX-alpha application, individual results depicted as in B (details in the main text). ( E ) Mean maximum rebound frequency, the firing rate over the first half second after the step (as in the examples in the inset, see methods for details, “maximum rebound frequency under control (red) and during DTX application (blue), and individual results depicted as in ( B ) (see main text for details).

Article Snippet: The specific Kv1 channel blockers: αDendrotoxin (DTX-alpha), DTX-I and DTX-K (100 nM) were diluted as recommend by the providers (Alomone, Latoxan) to prepare aliquots that were frozen and diluted to the final concentration in ACSF containing neurotransmitter blockers before use.

Techniques: Activity Assay, Injection, Blocking Assay

Journal: Scientific Reports

Article Title: Kv1 potassium channels control action potential firing of putative GABAergic deep cerebellar nuclear neurons

doi: 10.1038/s41598-020-63583-7

Figure Lengend Snippet: The action potential (AP) waveform of GAD + DCNs is sensitive to DTX-alpha application. ( A ) From left to right: Typical example of averaged spontaneous APs recorded from one GAD + DCN during application of neurotransmitter blockers (control), during DTX-alpha (100 nM) application (DTX-alpha), and after correcting the ISI Vm by current injection during DTX-alpha application (DTX-alpha& DC), and all traces superimposed. The insets depict in expanded scale the control and DTX-alpha traces. The first one illustrates the hyperpolarization during the ISI (marked by the arrow). The second one, the different voltage threshold (noted by arrows). ( B ) Phase plots corresponding to the AP traces in A (the derivative of the Vm as a function of the Vm). B’-Detail of B in expanded scale showing the shift in AP voltage threshold (indicated by the horizontal dashed line, see methods) from control (red vertical dashed line) to more hyperpolarized levels during DTX, even after matching the ISI-Vm (green vertical dashed line). ( C ) Mean AP threshold for all recorded GAD + DCNs under control (red bar) and DTX-alpha application (ISI-Vm matching control levels, green bar) and individual results (depicted by the gray lines). Error lines indicate here and in the next figures SEM. ( D ) Mean AP HW for all recorded GAD + DCNs under control (red dot) and DTX-alpha plus DC (green dot), with individual results depicted as in ( C ). ( E ) Mean AP repolarizing rate (minimum dV/dt) for all recorded GAD + DCNs under control (red) and DTX-alpha plus DC (green), with individual results depicted as in ( C ). ( F ) Mean minimum ISI-membrane potential (ISI-Vm) for all recorded GAD + DCNs under control (red) and DTX-alpha plus DC (green), with individual results as in ( C ).

Article Snippet: The specific Kv1 channel blockers: αDendrotoxin (DTX-alpha), DTX-I and DTX-K (100 nM) were diluted as recommend by the providers (Alomone, Latoxan) to prepare aliquots that were frozen and diluted to the final concentration in ACSF containing neurotransmitter blockers before use.

Techniques: Injection

Journal: Scientific Reports

Article Title: Kv1 potassium channels control action potential firing of putative GABAergic deep cerebellar nuclear neurons

doi: 10.1038/s41598-020-63583-7

Figure Lengend Snippet: Kv1 channels control the spiking activity and membrane potential of silent and spontaneously active GAD + DCNs. ( A ) Mean spontaneous frequency under control and DTX conditions (data from DTX-alpha, DTX-I and DTX-K together, no DC applied, see details in main text). ( B ) CV of spontaneous ISIs under control and DTX conditions (box illustrates 10 and 90% percentiles and whiskers the error, see details in main text). ( C ) DTX effect on a previously silent DCN (DTX-alpha, 100 nM, time of application indicated by the bar on top), illustrates the depolarization and beginning of tonic firing evoked by DTX. The inset illustrates in expanded time base the beginning of the tonic discharge. (For clarity the responses to short pulses (10 ms) applied every two seconds before spiking started were digitally removed and substituted by a straight line). ( D ) Two examples of GAD + DCN recorded using 25 KHz sampling rate, illustrate typical DTX effects on averaged spontaneous APs. Top, in this example DTX induced a depolarization of the ISI-Vm. After DC injection to match control ISI-Vm (green trace), the spike width was narrower than control (HW: 0.59 and 0.56 for control and DTX&DC respectively). Bottom, in this example, the ISI-Vm hyperpolarized and the AP duration increased during DTX (HW: 0.49 and 0.54 ms for control and DTX respectively). ( E ) Scatter plot of DTX induced changes in HW (ms) as a function of the changes induced in ISI-VM (mV, note that most neurons that hyperpolarized displayed elongations in HW).

Article Snippet: The specific Kv1 channel blockers: αDendrotoxin (DTX-alpha), DTX-I and DTX-K (100 nM) were diluted as recommend by the providers (Alomone, Latoxan) to prepare aliquots that were frozen and diluted to the final concentration in ACSF containing neurotransmitter blockers before use.

Techniques: Activity Assay, Sampling, Injection

Journal: Scientific Reports

Article Title: Kv1 potassium channels control action potential firing of putative GABAergic deep cerebellar nuclear neurons

doi: 10.1038/s41598-020-63583-7

Figure Lengend Snippet: The action potential waveform of non-GAD + DCNs, putative glutamatergic principal DCNs is sensitive to DTX. ( A ) From left to right: Typical example of averaged spontaneous action potentials recorded from a large non-GAD + DCN during application of neurotransmitter blockers (control, red), during DTX-alpha (100 nM) application (DTX-alpha, blue), and after matching the ISI Vm to control levels by current injection during DTX-alpha application (DTX-alpha& DC, green), and all traces superimposed. ( B ) The corresponding phase plots of the traces illustrated in A. ( C ) Mean AP threshold before and during DTX application for putative glutamatergic principal DCNs and individual results depicted by the gray lines (see details in main text). ( D ) Mean AP HW before and during DTX application for putative glutamatergic principal DCNs and individual results depicted as gray lines (see main text for details). ( E ) Mean AP repolarizing rate (minimum dV/dt) before and during DTX application for putative glutamatergic principal DCNs and individual results as gray lines (see details in main text).

Article Snippet: The specific Kv1 channel blockers: αDendrotoxin (DTX-alpha), DTX-I and DTX-K (100 nM) were diluted as recommend by the providers (Alomone, Latoxan) to prepare aliquots that were frozen and diluted to the final concentration in ACSF containing neurotransmitter blockers before use.

Techniques: Injection

Journal: bioRxiv

Article Title: Kv1 potassium channels control action potential firing of putative GABAergic deep cerebellar nuclear neurons

doi: 10.1101/779082

Figure Lengend Snippet: A- From left to right: The traces illustrate typical example of spontaneous action potentials and the dots on top of each spike their corresponding instantaneous frequency using the same scale on the left recorded from one GAD+DCN during application of NT blockers (control), during DTX-alpha (100nM) application (DTX-alpha), application of NT blockers (control), during DTX-alpha (100nM) application (DTX-alpha), and after correcting the ISI Vm by current injection during DTX-alpha application (DTX-alpha& DC) (For comparison, same neuron as in ). B- Mean spontaneous frequency of GAD+DCNs before (control) and during DTX-alpha application (see main text for details). B’- Mean change in spontaneous frequency induced by DTX-alpha application and individual results superimposed (see main text for details). C- Top: left traces illustrate from the typical changes in spiking activity induced by injection of depolarizing current pulses of 1 second duration and increasing current intensity in one GAD+DCN recorded under control conditions used to determine the maximum repetitive frequency (MRF, see methods for details). MRF is determined by measuring the frequency at steady state (rectangle) just before the step inducing depolarizing block (top trace). Bottom left: Mean MRF for GAD+DCNs before (control) and during DTX-alpha application (see main text for details). Top right: The trace illustrates the response induced by injection of hyperpolarizing current pulses of 1 second duration in one GAD+DCN recorded under control conditions. The hyperpolarization typically causes increases in firing rate respect to basal conditions after the hyperpolarization (rebound response). The maximum firing rate over the first second after the step (see methods for details, rectangle, “Maximum Rebound frequency”), was used to characterize changes in the rebound response. Bottom right: Mean Maximum Rebound frequency for GAD+DCNs before (control) and during DTX-alpha application (see main text for details).

Article Snippet: Dendrotoxins DTX-alpha, DTX-I and DTX-K (100nM) were diluted as recommend by the providers (Alomone, Latoxan) to prepare aliquots that were frozen and diluted to the final concentration in ACSF containing neurotransmitter blockers before use.

Techniques: Injection, Activity Assay, Blocking Assay

Journal: bioRxiv

Article Title: Kv1 potassium channels control action potential firing of putative GABAergic deep cerebellar nuclear neurons

doi: 10.1101/779082

Figure Lengend Snippet: A- From left to right: Typical example of averaged spontaneous action potentials recorded from one GAD+DCN during application of NT blockers (control), during DTX-alpha (100nM) application (DTX-alpha), and after correcting the ISI Vm by current injection during DTX-alpha application (DTX-alpha& DC), and all traces superimposed. The inset depicts in expanded scale the control and DTX-alpha traces. B- Phase plots corresponding to the traces in A (the derivative of the Vm as a function of the Vm). B’-Detail of B in expanded time scale to show the hyperpolarizing shift in the rapid raise in dV/dt signaling the AP threshold. C- Mean change in AP threshold for all recorded GAD+DCNs under DTX-alpha and individual results superimposed (some data points overlap). D- Mean change in AP HW for all recorded GAD+DCNs under DTX-alpha and individual results superimposed. E- Mean change in the AP repolarizing rate (minimum dV/dt) for all recorded GAD+DCNs under DTX-alpha and individual results superimposed. F- Mean change in minimum ISI-membrane potential (ISI_Vm) for all recorded GAD+DCNs under DTX-alpha and individual results superimposed.

Article Snippet: Dendrotoxins DTX-alpha, DTX-I and DTX-K (100nM) were diluted as recommend by the providers (Alomone, Latoxan) to prepare aliquots that were frozen and diluted to the final concentration in ACSF containing neurotransmitter blockers before use.

Techniques: Injection

Journal: bioRxiv

Article Title: Kv1 potassium channels control action potential firing of putative GABAergic deep cerebellar nuclear neurons

doi: 10.1101/779082

Figure Lengend Snippet: A- Traces illustrate recordings from one silent DCN before (control) and during application of 4-AP 15μM. Note the depolarization and spiking activity. B- Averaged APs recorded before and during 4-AP application from the same neuron. C- Phase plot corresponding to the traces shown in B. Note the shift in threshold and remarked decrease in minimum dV/dt. D- Traces illustrate recordings from one silent GAD+DCN before (control) and during application of DTX-alpha (100 nM). E- Averaged APs recorded before and during DTX-alpha application from the same neuron as in D. F- Phase plots corresponding to the traces in E.

Article Snippet: Dendrotoxins DTX-alpha, DTX-I and DTX-K (100nM) were diluted as recommend by the providers (Alomone, Latoxan) to prepare aliquots that were frozen and diluted to the final concentration in ACSF containing neurotransmitter blockers before use.

Techniques: Activity Assay

Journal: bioRxiv

Article Title: Kv1 potassium channels control action potential firing of putative GABAergic deep cerebellar nuclear neurons

doi: 10.1101/779082

Figure Lengend Snippet: A- From left to right: Typical example of averaged spontaneous action potentials recorded from a large Non-GAD+DCN during application of NT blockers (control), during DTX-alpha (100 nM) application (DTX-alpha), and after matching the ISI Vm to control levels by current injection during DTX-alpha application (DTX-alpha& DC), and all traces superimposed. B- The corresponding phase plots of the traces illustrated in A. C- Mean AP threshold before and during DTX application for putative glutamatergic principal DCNs (see text for details). D- Mean AP HW before and during DTX application for putative glutamatergic principal DCNs (see text for details). E- Mean AP repolarizing rate (minimum dV/dt) before and during DTX application for putative glutamatergic principal DCNs (see text for details).

Article Snippet: Dendrotoxins DTX-alpha, DTX-I and DTX-K (100nM) were diluted as recommend by the providers (Alomone, Latoxan) to prepare aliquots that were frozen and diluted to the final concentration in ACSF containing neurotransmitter blockers before use.

Techniques: Injection

Journal: eNeuro

Article Title: Identification of Persistent and Resurgent Sodium Currents in Spiral Ganglion Neurons Cultured from the Mouse Cochlea

doi: 10.1523/ENEURO.0303-17.2017

Figure Lengend Snippet: TTX-sensitive transient and persistent Na + currents identified in SGNs cultured from hearing mice. A , Current responses recorded using K + -filled patch electrodes, from a representative rapidly-adapting P14 SGN during 200-ms voltage steps. A transient inward Na + current (I NaT ) was followed by LVA and HVA outward K + currents. B , Current response from the same cell during a voltage ramp (between −143 and +47 mV, duration 400 ms) before (control) and after bath application of 100 nM DTX-K. DTX-K blocked the LVA K + current, revealing a small inward current activated at potentials positive to −65 mV (detailed in the inset). C , Current responses recorded using K + -filled patch electrodes, from a representative nonadapting P14 SGN during 200-ms voltage steps (protocol described in A ). Activation of I NaT was followed by activation of HVA K + currents. LVA K + currents were absent from this cell. D , Current responses from the same SGN, during a voltage ramp before (control) and after bath application of 100 nM TTX. A TTX-sensitive inward current activated at potentials positive to −65 mV (difference current, detailed in the inset). E , TTX-subtracted currents recorded from the SGN in D , during 200-ms depolarizing voltage steps. A voltage step to −53 mV activated a small persistent inward current (I NaP ), whereas the voltage step to −33 mV activated a large I NaT ( * , peak current cropped for clarity) and a small I NaP . F , Comparison of the voltage dependence of TTX-sensitive I NaT and I NaP (measured at the arrow in E ) in this SGN.

Article Snippet: Dendrotoxin-K (DTX-K), TTX, 4,9-anhydro-TTX (4,9-ah-TTX), and sea anemone ( Anemonia sulcata ) toxin ATX-II were all obtained from Alomone Labs. Toxins were prepared as stock solutions in water and stored at −20°C, before dilution in extracellular solution for patch clamp recordings.

Techniques: Cell Culture, Activation Assay