What is the dynamic clamp?       Some of the differences between using windows based DC is that the achieved update rates of 2-20 KHZ, this is fast enough for most purposes but extremly fast conductacnes as Na currents can not be approximated very well, other is that windows distributes processor time between different tasks so can lead to discontinuties and gaps in the DC process and prevent real time performance or low update of rates. This implememtention in windows is free and only requires the standard electrophisiology rig. Linux, you don’t have the interuption problem as window and can achieve rates of 20- 50 KH. It requieres expertise to be able to installed and operated the real time operating system. Accurate dynamic-clamp performance requires uninterrupted, rapid sampling of the membrane potential and fast computation of the current to be injected.

Applications of the dynamic clamp Simulating voltage-independent conductances The applications were divides in 5 broad categories: Simulation of voltage-indenpedent conductances, it is the simplest things thatc an be achieved with DC and its is useful to study the effects of leakege conducatnces of ligand conductacnes of nueronal dynamics. The DC conducatnces act with the normal membrane conductances of the cell and the interection between them, the added/substracted conductance and the exiting conductances is what makes the manipulations interesting. The substraction will simulate a negative conductance designed to cancel out the of the leakeage introduced by the electrode penetration. Adding or subtracting voltaged-gated conductances

Applications of the dynamic clamp Simulating synapses Simulating synapes, the clamped neuron acts as the postsynaptic cell and another neuron or computer model acts as the source of the presynaptic input. This approached requieres record the membrane potential or the presynaptic cell and using DC to cntrol the current injection into the postsynaptic cell. A synpase in the circuit can be incrase or reduce in order to study its effect on the network activity or simulated synapese can be introduced where none exixted before and study completely novel neural circuits Couplig to model neurons, we can construt hybrid circuits that invlove the interacting computer modeled and biological elelments. Create networks. Coupling to model neurons

Applications of the dynamic clamp Simulating in vivo conditions Simulating in vivo conditions, some of the disavantages of studying slice preparations is that you have les synaptic input, it is a different enviorment for the cell, can be relativily silent. So the DC offers a way to studying the neurons in slice preparations while simuating the in vivo synaptic input.

Michael N. Economo, Fernando R. Fernandez, and John A. White Dynamic Clamp: Alteration of Response Properties and Creation of Virtual Realities in Neurophysiology Michael N. Economo, Fernando R. Fernandez, and John A. White

Applications of Dynamic Clamp Applying a certain type of current. Modeling a synapse. Modeling a whole cell. Role of cells in a network. Feedback mechanisms. Creating in vivo like conditions. Hybrid systems.

Importance of stochasticity of INaP in sub-threshold oscillations stellate cells. (Dorval and White 2005) Sub-threshold oscillations of INaP is the source of membrane potential noise. This is resulted from relatively small number of NaP channels with high single channel conductance. They block INaP and inject current with dynamic clamp. They inject current in a stoschastic and deterministic way and compare difference.

Producing in vivo like conditions (Destexhe et al., 2003) Neurons in the nervous system receives a lot of synaptic input. This inputs reduces input resistance and time constants by 80% nad provide a huge depolarization. Dynamic clamp is used to provide in vivo like conditions in vitro by applying high rates of artificial inputs.

Hybrid Networks A real cell can be connected to one or more in silico artificial cells. Synchronization between reciprocally connected cells in a network can be studied. Could be used for model validation. Model can be tested in vitro with a real cell. Spike time differences between a stellate cell and an artificial cell simulated. Different virtual connections used as shown in figure. Different patterns of synchronized activity recorded. (Netoff et al., 2005)

Feedback control Dynamic clamp is used to knock-in conductances which have no physiological correlate. After-hyperpolarization (AHP) reducing and enhancing currents injected. It is shown that spike train rhythmicity is determined by the shape of AHP. These currents manipulate the voltage trajectory following a spike. Entorhinal stellate cells (Fernandez and White 2008)

Dynamic-clamp: Limitations Hardware / software capacity and price Conversion between analog and digital signals Time error/ frequency rate Conductances simulated are restricted to the site of injection Duplicates the electrical but not the signal conductances consequences Voltage measurement errors (single/double pipette; access resistance) Model error Traditional errors Accurate dynamic-clamp performance requires uninterrupted, rapid sampling of the membrane potential and fast computation of the current to be injected. If the sampling and computation are fast enough, the electrophysiological effects of any ion-conducting channels can be reproduced as if these were located at the site of voltage measurement and current injection. A major limitation of the dynamic clamp is that the conductances it simulates are restricted to the site of current injection. As a result, conductances located far from the injection site can be mimicked only approximately. The dynamic clamp duplicates the electrical but not the signal conductances consequences elicited by specific ionic currents. In particular, with conventional electrode solutions, the dynamic clamp can simulate the electrical current from a set of Ca++ channels, but it doesn’t reproduce the changes in intracellular Ca++ concentration that normally accompany the gating of each channel. Other limitations are shared by traditional current and voltage-clamp techniques: artifacts of electrode resistance and capacitance. These artifacts can be minimized by using low-resistance electrodes, by using separate electrodes for voltage recording and current injection, or by temporally separating recording and injection through a single electrode using the discontinuous current-clamp technique.

What is the role of NaV channels in SN DA neuron activity? Introduction Substantia Nigra (SN) dopamine (DA) neurons exhibit slow intrinsic pacemaker activity Voltage-gated sodium channels (Nav) contribute to the slow depolarization (DP) phase leading to action potential (AP) initiation in the axon initial segment (AIS) and propagation. DP block is preceded by attenuation of AP, amplitude, broadening of each successive spike, and the eventual failure of AP production Substantia nigra (SN) dopamine (DA) neurons exhibit slow intrinsic pacemaker activity. Although voltage-gated calcium channels have been implicated in driving pacemaker activity voltage-gated sodium (NaV) channels also contribute to the slow depolarization (DP) phase leading to action potential (AP) initiation. Furthermore, NaV channels are responsible for the initiation of APs in the axon initial segment (AIS) and their subsequent propagation in the axon, dendrites, and soma, which in turn triggers DA release from terminals and dendrites. Thus, NaV channels are major determinants of nigral DA neuron activity and function. All antipsychotic drugs (APDs) in use block dopamine (DA) D2 receptors and these drugs are most effective at alleviating the psychotic features of schizophrenia . After3+ weeks of treatment, DA neurons undergo depolarization block, decreasing the number of DA neurons firing spontaneously due to overexcitation and spike inactivation. All drugs that are therapeutically effective cause depolarization block in the mesolimbic DA neurons, whereas those that cause extrapyramidal side effects induce depolarization block of the nigrostriatal DA neurons, which will alleviate psychotic symptoms such those that occur during schizophrenia. Interestingly, the efficacy of antipsychotic drugs is correlated with induction of chronic DP block in DA neurons What is the role of NaV channels in SN DA neuron activity?

Sodium channels and SN DA neuron model description http://neuromorpho.org/neuroMorpho/rotatingImages/DAN-04-R.CNG.gif Here the role of NaV channels in SN DA neuron activity is investigated by manipulating NaV current magnitude, distribution, and gating with the dynamic clamp, a device that adds virtual channels via computationally generated currents injected through a patch pipette into the neuron. Specifically, virtual NaV conductance, which was based on native SN DA neuron currents characterized in nucleated patches (Seutin and Engel, 2010). Even though APs initiate in the AIS, somatic NaV channels are shown to control the balance between pacemaker frequency and susceptibility to DP block in nigral DA neurons. A) Comparison of the peak NaV current/voltage response measured from SN DA neuron nucleated patches by Seutin and Engel (2010) (circles) and the modeled NaV current/voltage response based on fitting those data (squares and line). The description of the NaV current kinetics used in both the modeling and dynamic clamp experiments was derived from rat SN DA nucleated patch voltage-clamp recordings published by Seutin and Engel (2010). B) Steady-state inactivation (h; solid line) and activation (m 3 ; dashed line) curves of the model NaV current used in both dynamic clamp and modeling experiments. This figure shows the resulting voltage dependence of steady-state activation and inactivation for the modeled/virtual NaV channel. C) On the left: The SN DA neuron model was implemented in the NEURON simulation package (Hines and Carnavale, 1997) using a real DA neuron morphology (Vetter et al., 2001) downloaded from the neuromorpho. org database. C) On the right: The simulated membrane currents included: (1) the constitutively active GIRK conductance (Bradaia et al., 2009) and a sodium leak (Khaliq and Bean, 2010), which contribute to the net background current (Ib), (2) an L-type calcium conductance (ICav,L) (Durante et al., 2004), (3) a composite potassium conductance (IKv) (Ding et al., 2011), and (4) the TTX-sensitive sodium conductance (INav) (Seutin and Engel, 2010)

Inward currents of the AIS and soma are reduced during DP block induction To induce DP block, SN DA neurons were current-clamped to 60 mV and then depolarized by either a 250 pA current injection. Each stimulus induced APs that attenuated progressively (i.e., peak values dropped while AHPs and thresholds became depolarized) until activity ceased. Current ( I) flowing during these responses is evident from dV/dt because dV/dt I/Cm, where Cm equals membrane capacitance. Specifically, because APs in DA neurons initiate in the AIS and backpropagate into the soma, plotting dV/dt versus time or membrane potential reveals two inward current components during the upstroke of the AP: initial activation of NaV channels in the AIS and subsequent NaV channel activation in the soma. Both the AIS and somatic NaV currents revealed by dV/dt plots progressively decreased. The arrows indicate the peak net current flowing during the AIS and somatic portions of the AP. An upward deflection indicates a net inward current and a downward deflection indicates a net outward current. On the right is a phase plane plot, usually describes 2 state variable systems , so it consists of a curve of one state variable plotted against another one. The flows in the vector field indicate the time evolution. dV/dt = I/Cm

Inward currents of the AIS and soma are reduced during DP block induction 250 pA injection – n = 9 30 nS gNMDA injection – n = 7 Ok so now that we are familiar with the traces used throughout the experments, I have the last slide on the left. On the right, the same protocole was used but this is the case of a simulated experiment, so instead of injecting a current to trigger depolarization, the authors are virtually injecting 30 nS of NMDAR by dynamic clamp. So NMDAR is the glutamate receptor, its ionotropic , and binding of the ligand to this receptor results in the opening of non selective cation channels.

Reduction of AIS and somatic Nav current increases susceptibility to DP block To determine whether the reduction of NaV channel activity is sufficient to increase susceptibility to DP block, a submaximal concentration (10 nM) of TTX was bath applied to the slice and SN DA neurons were depolarized with small current injections.. Representative APs (top) from a SN pars compacta DA neuron current-clamp recording in response to a 75 pA current injection before (control) and 20 min after 10 nM TTX application (10 nM TTX) and their corresponding dV/dt (middle) and phase plane (bottom) plots. So if we compare the Aps, adding 10 nM TTX resulted in a more depolarized threshold and a slower rise time. Submaximal concentration = partial block dV/dt plots revealed that TTX reduced both the AIS and somatic NaV currents by one third. Figure B, top: representative current clap recording in response to a 5s 75 pA step dring normal pacing. Down: blocking Nav induced a DP block. Therefore, the authors conclude that global partial inhibition of NaV channels increases the susceptibility of SN DA neurons to DP block.

Addition of somatic Nav current decreases susceptibility to DP block The contribution of somatic NaV channels to DP block was then examined by adding virtual NaV channels with the dynamic clamp to the soma (i.e., the position of the patch pipette passing current from the dynamic clamp). After 20 min of 10 nM TTX application, somatic virtual NaV channels altered the first AP evoked by a 75 pA current injection in current clamped neurons: addition of somatic virtual NaV channels increased AP height and the somatic component of the dV/dt plot without changing threshold or the AIS component of the dV/dt plots. In this example in A, addition of 100 nS of virtual NaV conductance nearly replaced the native NaV channels blocked by TTX in the soma, but had no effect in the AIS. Strikingly, this soma-specific manipulation prevented DP block in this neuron (compare Figs. 3B, 4B). The reduction in susceptibility to DP block was evident with a variety of current injections in independent experiments. Furthermore, while 100 nS of somatic virtual NaV channels delayed DP block, 200 nS prevented DP block. Therefore, the increased sensitivity to DP block produced by pharmacological reduction of NaV channel activity in both the soma and AIS is reversed by adding NaV channels solely to the soma.

Addition of virtual anti-Nav channels to the soma hastens to DP block Dynamic clamp can also be used to add virtual anti-NaV channels to counterbalance the activity of native channels in the soma. In these experiments, native somatic channels respond normally to changes in membrane potential, but their depolarizing currents are nullified by current from virtual anti-channels that gate normally, but produce current that flows in the opposite direction. Thus, with this approach, the dynamic clamp produces a net reduction in somatic NaV conductance. The spatial specificity of virtual anti-NaV channels was apparent from the AP waveform and dV/dt plots (Fig. 6A): only the somatic component was inhibited by the antichannels. Reducing the somatic NaV current in this way hastened DP block. The dynamic clamp technique was used to add 100 nS of virtual anti-NaV conductance to the soma of an SN DA neuron while being current-clamped to60mVfollowed by a 250 or 300 pA current step or 30 nS virtual NMDA conductance. In A, Representative traces of the first AP evoked by 250 pA current injection (top) under control conditions (control) and 100 nS of virtual anti-NaV channel conductance (100 nS gNaV) and their corresponding dV/dt (middle) and phase plane (bottom) plots. Together, the responses to virtual NaV channels and anti-NaV channels (Figs. 5, 6) demonstrate that even though APs initiate in the AIS, somatic NaV channel density controls the susceptibility to DP block.

Replacing native Nav with virtual Nav reconstitutes pacing at a higher frequency The marked effects of somatic NaV channels in the presence of native AIS and dendritic NaV channels raise the question of whether somatic NaV channels alone are sufficient for normal DA neuron pacemaker activity. To address this issue, all native NaV channels and APs were blocked with 1 M TTX while current clamping neurons to 60 mV. Then 2 s long 50 pA current injections were applied to elicit DP (Fig. 8A). Finally, various levels of virtual NaV conductance were added back to the soma with the dynamic clamp (Fig. 8A, bottom two traces) until the height of the first AP (measured from peak to trough of AHP) began to approach control levels (Fig. 8A,B). A, Representative SN DA neuron current-clamp recording held at60 mV followed by a 2 s long, 50 pA current injection before (pre-TTX; top trace) and after maximal block of TTX-sensitive sodium channels with 1M TTX (second trace) followed by application of 600 nS (third trace) and 800 nS (fourth trace) of virtual NaV conductance to the soma. B, AP height and (C) pacing frequency before TTX application (Native) and after 10 min of 1M TTX application with different levels of virtual NaV conductance With the production of large APs, repetitive spiking was evoked with NaV channels only in the soma (Fig. 8A,D). However, this activity was abnormally fast; for example, 800 nS of virtual NaV conductance, which produced APs that were 80% of the control height (Fig. 8B), resulted in pacemaker-like activity that was3-fold faster than that of the native distributed channel (Fig. 8C). Furthermore, somatic virtual channels reconstituted true pacemaker activity (i.e., repetitive spiking without added bias current injection), but spiking frequency was again higher than normal (Fig. 8D; n 11). The production of abnormally high pacemaker rates by somatic restriction of NaV channels implies that the widespread distribution of NaV channels in the somatodendritic compartment of SN DA neurons is critical for the production of native slow pacemaker activity.

Simulation predictions of frequency with differential somatodendritic Nav channel distribution The experiments thus far examined the effect of changing NaV channel density in the soma and eliminating NaV channels in dendrites. However, because DA neurons possess multiple dendrites, it is not possible to alter NaV channel density uniformly and simultaneously throughout the dendritic compartment with the dynamic clamp. However, the above analysis demonstrated the predictive power of modeling, in which it is possible to control compartmental channel distribution. Therefore, simulations were used to vary the balance of somatic and dendritic NaV channels to understand how distribution affects spiking frequency in DA neurons. Specifically, recordings of responses to 80 pA injections into the soma of neurons current-clamped to 60 mV were simulated with varying distributions of NaV channels in the soma and dendrites while keeping the total across both compartments a constant. Strikingly, as gNaV is moved from the soma to the dendrites, spiking frequency decreases (Fig. 12A,B). The plot of frequency dependence in Figure 12B shows that the uniform distribution found in native DA neurons produces a minimal pacemaker rate, while preserving somatic channels. In SN DA neurons, the preservation of somatic channels is critical for reliable backpropagation of APs through the soma into other nonaxon-baring dendrites and concomitant release of DA. Modeling was also used to determine the effect of adding gNaV to different compartments in neurons with an initially uniform distribution of NaV channels (i.e., as found in native neurons). Strikingly, although the dendritic compartment is far larger than the soma, adding extra channels to the soma alone is as effective in increasing pacemaker frequency as distributing the extra channels throughout the dendrites or the whole somatodendritic compartment (Fig. 12C). This finding further emphasizes the critical role of NaV channels in nigral DA neuron pacemaker frequency.

Rat somatotrophs and lactotrophs -> bursting Gonadotrophs -> spiking Difference due BK current Rat somatotrophs and lactotrophs exhibit spontaneous bursting and have high basal levels of hormone secretion, while gonadotrophs exhibit spontaneous spiking and have low basal hormone secretion. It has been proposed that the difference in electrical activity between bursting somatotrophs and spiking gonadotrophs is due to the presence of large conductance potassium (BK) channels on somatotrophs but not on gonadotrophs. This is one example where the role of an ion channel type may be clearly established. So the test this with DC

MODEL Membrane potential   Membrane potential             It has a stochastic current that represent a channel noise to create irregular volatge time courses as observed in pitutary cells. Some of the assumption that they made for the model, are that the Bk channels are located near the Ca channels. The Ca concentration in nanodomains equilibrated within seconds then they modeled the gating of BK channels only dependet in voltage            

Model Predictions Burstiness was compute using a distribution of event duration. The burtiness factor is define as the fraction of events that are burst. Check how change the burst when we add gBK. This results were with tau constant as 5 if we change it we can see that for bigger values we lost bursting therefore the BK channels must activate quickly to promote bursting

Random gK, gSK, gCa To demonstrate the robustness of these results to parameter choices, they ran 512 simulations for which the values of the conductances where chosen randomly between -50% and 50% of their default values. Fisrt 30% burst, last burst 80% From here we can see that the precesce of BK is no necesarry for busting but the majotrity of the models the precesnce /abcense of BK conductance predicts the occurance/ absence of busrting.

Pharmacology blockage and Dynamic Clamp If a fast voltage-dependent BK conductance is responsible for bursting in pituitary cells, then blocking BK channels should abolish bursting. Moreover, bursting should be restored by adding back an artificial, fast voltage-dependent outward conductance via dynamic clamp. Lacto-somatrotroph cells genreate spiking, busrting, or mixed firing. They blocked BK with paxiline or iberiotoxin., it converte the bursting cells to spiking cells (11/13 stop bursting circle 2/2 just spike before and after triangle ) adding BK with DC it change remove the effect of the drug and the spiking activity change to bursting. The parameter use in the model of DC were selected to approximatley match the current volatge curves obtained with votage-clamp recordings. Most cells required a maximal BK conductance of 0.5nS to switch to a bursting pattern. Increasing the conductance of the injected BK current also increased burst duration. The conversion of spikers to bursters after the DC suggest that the difference in activity pattern is explain at least in a part due the difference in BK conducatnce.

-gBK The if we artificially subtracting a fast BK conductance from bursting cells in control conditions should switch the activity of these cells to spiking. 6/7 cell acts similar as pharmacology blakcage. And the addition addition of a fast BK conductance in control conditions increased burstiness to higher than control values. These effects were usually more increase with higher levels of gBK injection. So these results suggest that higher density of fast-activating BK conductance leads to higher burstiness

Higher density of fast-activating gBK -> higher burstiness BK currents recorded in response to voltage steps, averaged over four steps from a holding V =- 40 to 0, 10, 20, and 30 mV. The voltage steps were performed before and after pharmacological BK block, and the difference current was computed. The two traces correspond to two different cells,one bursting in control conditions(*), one spiking( +) This supports the hypothesis that bursters have higher levels of fast-activating BK conductance than spikers. These results also indicate that the fast component of the activated BK current is important for bursting, while the total amount of BK current that slowly increases with time is not.

Activation time constant How the pitutary cell generate the burstig? Hypotesis is that the fast activation of BK channels limits spike amplitude and prevents full activation of delayed-rectifier K current, preventing full repolarization and allowing membrane potential to remain at a depolarized level, until the event is terminated by activation of SK channels. If this is true, then adding an artificial BK conductance with a slower time constant to cells with low burstiness should not increase burstiness as efficiently as a faster-activating BK conductance. So they change the time constant.

Spiking gonadotrophs to bursters Artificialfast-activatingBKcurrentpromotesburstingingonadotrophs

Conclusions gBK Time constant for BK

Sources http://www.hormone.org/questions-and-answers/2010/hyperprolactinemia

Transient outward K+ current reduction prolongs APs and promotes after depolarizations: a dynamic clamp study in human and rabbit cardiac atrial myocytes. A. J. Workman, G. E. Marshall, A. C. Rankin, G. L. Smith and J. Dempster

Key Points The effects of a transient outward K+ current (ITO) on AP shape and duration in atrial myocytes are investigated. Dynamic clamp is used for blocking this current, since ITO blocking drugs are non-selective. (For example; 4-AP blocks IKur with a more than 40 times greater affinity than ITO)

Computational Model

Modeling ITO

Activation, Inactivation and Time Constants

Rabbit Myocytes: AP Response to ITO Subtraction

APD

Human Myocytes: AP Response to ITO Subtraction

APD

Effect of ITO on Different Phases (Human) (ITO Subtraction Interrupted at indicated time points)

CADs : Cellular Arrhythmic Depolarization Evidence for Delayed After Depolarization (DAD) (Rabbit)

Modulating CADs with ISO and ITO Subtraction

Within Train CADs

Production of Early After Depolarizations (EADs) by ITO Subtraction and β-Stimulation

Suppression of CADs by interrupting ITO Subtraction, Adding ITO and β1-Antogonist

CONCLUSION Dynamic clamp can be used to better understand the roles of specific currents. Dynamic Clamp is used to neutralize a transient outward current by injecting current at opposite direction. They showed the importance of a transient outward K+ current on normal heart functioning.