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The resurgent sodium current (INaR) activates on membrane repolarization, such as during the downstroke of neuronal action potentials. Due to its unique activation properties, INaR is thought to drive high rates of repetitive neuronal firing. However, INaR is often studied in combination with the persistent or noninactivating portion of sodium currents (INaP). We used dynamic clamp to test how INaR and INaP individually affect repetitive firing in adult cerebellar Purkinje neurons from male and female mice. We learned INaR does not scale repetitive firing rates due to its rapid decay at subthreshold voltages and that subthreshold INaP is critical in regulating neuronal firing rate. Adjustments to the voltage-gated sodium conductance model used in these studies revealed INaP and INaR can be inversely scaled by adjusting occupancy in the slow-inactivated kinetic state. Together with additional dynamic clamp experiments, these data suggest the regulation of sodium channel slow inactivation can fine-tune INaP and Purkinje neuron repetitive firing rates. [ABSTRACT FROM AUTHOR]

Loss-of-function variants in the gene SCN2A, which encodes the sodium channel NaV1.2, are strongly associated with autism spectrum disorder and intellectual disability. An estimated 20%-30% of children with these variants also suffer from epilepsy, with altered neuronal activity originating in neocortex, a region where NaV1.2 channels are expressed predominantly in excitatory pyramidal cells. This is paradoxical, as sodium channel loss in excitatory cells would be expected to dampen neocortical activity rather than promote seizure. Here, we examined pyramidal neurons lacking NaV1.2 channels and found that they were intrinsically hyperexcitable, firing high-frequency bursts of action potentials (APs) despite decrements in AP size and speed. Compartmental modeling and dynamic-clamp recordings revealed that NaV1.2 loss prevented potassium channels from properly repolarizing neurons between APs, increasing overall excitability by allowing neurons to reach threshold for subsequent APs more rapidly. This cell-intrinsic mechanism may, therefore, account for why SCN2A loss-of-function can paradoxically promote seizure.

The article discusses the role of the atypical sodium channel NALCN in regulating the resting membrane potential and spontaneous excitability of native mouse anterior pituitary endocrine cells. The study reveals that NALCN is a key determinant of the depolarized membrane potential in these cells. By using a shRNA-knockdown strategy, researchers found that NALCN plays a crucial role in controlling electrical activity and intracellular calcium transients in these cells. The study raises questions about the functional expression and regulation of NALCN in different cell types and its potential impact on hormone secretion and other cellular processes in the anterior pituitary. [Extracted from the article]

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease in which motor neurons degenerate, resulting in muscle atrophy, paralysis, and fatality. Studies using mouse models of ALS indicate a protracted period of disease development with progressive motor neuron pathology, evident as early as embryonic and postnatal stages. Key missing information includes concomitant alterations in the sensorimotor circuit essential for normal development and function of the neuromuscular system. Leveraging unique brainstem circuitry, we show in vitro evidence for reflex circuit-specific postnatal abnormalities in the jaw proprioceptive sensory neurons in the well-studied SOD1G93A mouse. These include impaired and arrhythmic action potential burst discharge associated with a deficit in Nav1.6 Na+ channels. However, the mechanoreceptive and nociceptive trigeminal ganglion neurons and the visual sensory retinal ganglion neurons were resistant to excitability changes in age-matched SOD1G93A mice. Computational modeling of the observed disruption in sensory patterns predicted asynchronous self-sustained motor neuron discharge suggestive of imminent reflexive defects, such as muscle fasciculations in ALS. These results demonstrate a novel reflex circuit-specific proprioceptive sensory abnormality in ALS.SIGNIFICANCE STATEMENT Neurodegenerative diseases have prolonged periods of disease development and progression. Identifying early markers of vulnerability can therefore help devise better diagnostic and treatment strategies. In this study, we examined postnatal abnormalities in the electrical excitability of muscle spindle afferent proprioceptive neurons in the well-studied SOD1G93A mouse model for neurodegenerative motor neuron disease, amyotrophic lateral sclerosis. Our findings suggest that these proprioceptive sensory neurons are exclusively afflicted early in the disease process relative to sensory neurons of other modalities. Moreover, they presented Nav1.6 Na+ channel deficiency, which contributed to arrhythmic burst discharge. Such sensory arrhythmia could initiate reflexive defects, such as muscle fasciculations in amyotrophic lateral sclerosis, as suggested by our computational model.

Effective treatment of pain remains an unmet healthcare need that requires new and effective therapeutic approaches. NaV1.7 has been genetically and functionally validated as a mediator of pain. Preclinical studies of NaV1.7-selective blockers have shown limited success and translation to clinical studies has been limited. The degree of NaV1.7 channel blockade necessary to attenuate neuronal excitability and ameliorate pain is an unanswered question important for drug discovery. Here, we utilize dynamic clamp electrophysiology and induced pluripotent stem cell-derived sensory neurons (iPSC-SNs) to answer this question for inherited erythromelalgia, a pain disorder caused by gain-of-function mutations in Nav1.7. We show that dynamic clamp can produce hyperexcitability in iPSC-SNs associated with two different inherited erythromelalgia mutations, NaV1.7-S241T and NaV1.7-I848T. We further show that blockade of approximately 50% of NaV1.7 currents can reverse neuronal hyperexcitability to baseline levels. [ABSTRACT FROM AUTHOR]

Differing from other subtypes of inhibitory interneuron, chandelier or axo-axonic cells form depolarizing GABAergic synapses exclusively onto the axon initial segment (AIS) of targeted pyramidal cells (PCs). However, the debate whether these AIS-GABAergic inputs produce excitation or inhibition in neuronal processing is not resolved. Using realistic NEURON modeling and electrophysiological recording of cortical layer-5 PCs, we quantitatively demonstrate that the onset-timing of AIS-GABAergic input, relative to dendritic excitatory glutamatergic inputs, determines its bi-directional regulation of the efficacy of synaptic integration and spike generation in a PC. More specifically, AIS-GABAergic inputs promote the boosting effect of voltage-activated Na+ channels on summed synaptic excitation when they precede glutamatergic inputs by >15 ms, while for nearly concurrent excitatory inputs, they primarily produce a shunting inhibition at the AIS. Thus, our findings offer an integrative mechanism by which AIS-targeting interneurons exert sophisticated regulation of the input-output function in targeted PCs. [ABSTRACT FROM AUTHOR]

Dynamic clamp, a hybrid-computational-experimental technique that has been used to elucidate ionic mechanisms underlying cardiac electrophysiology, is emerging as a promising tool in the discovery of potential anti-arrhythmic targets and in pharmacological safety testing. Through the injection of computationally simulated conductances into isolated cardiomyocytes in a real-time continuous loop, dynamic clamp has greatly expanded the capabilities of patch clamp outside traditional static voltage and current protocols. Recent applications include fine manipulation of injected artificial conductances to identify promising drug targets in the prevention of arrhythmia and the direct testing of model-based hypotheses. Furthermore, dynamic clamp has been used to enhance existing experimental models by addressing their intrinsic limitations, which increased predictive power in identifying pro-arrhythmic pharmacological compounds. Here, we review the recent advances of the dynamic clamp technique in cardiac electrophysiology with a focus on its future role in the development of safety testing and discovery of anti-arrhythmic drugs.

• I A K+ currents suppress the response of rNST neurons to afferent stimulation. • GABAergic inhibition also suppresses afferent-induced responses. • GABAergic inhibition and I A appear to operate independently. Responses in the rostral (gustatory) nucleus of the solitary tract (rNST) are modified by synaptic interactions within the nucleus and the constitutive membrane properties of the neurons themselves. The potassium current I A is one potential source of modulation. In the caudal NST, projection neurons with I A show lower fidelity to afferent stimulation compared to cells without. We explored the role of an A-type K+ current (I A) in modulating the response to afferent stimulation and GABA-mediated inhibition in the rNST using whole cell patch clamp recording in transgenic mice that expressed channelrhodopsin (ChR2 H134R) in GABAergic neurons. The presence of I A was determined in current clamp and the response to electrical stimulation of afferent fibers in the solitary tract was assessed before and after treatment with the specific Kv4 channel blocker AmmTX3. Blocking I A significantly increased the response to afferent stimulation by 53%. Using dynamic clamp to create a synthetic I A conductance, we demonstrated a significant 14% decrease in responsiveness to afferent stimulation in cells lacking I A. Because I A reduced excitability and is hyperpolarization-sensitive, we examined whether I A contributed to the inhibition resulting from optogenetic release of GABA. Although blocking I A decreased the percent suppression induced by GABA, this effect was attributable to the increased responsiveness resulting from AmmTX3, not to a change in the absolute magnitude of suppression. We conclude that rNST responses to afferent input are regulated independently by I A and GABA. [ABSTRACT FROM AUTHOR]

Dynamic clamp, a hybrid-computational-experimental technique that has been used to elucidate ionic mechanisms underlying cardiac electrophysiology, is emerging as a promising tool in the discovery of potential anti-arrhythmic targets and in pharmacological safety testing. Through the injection of computationally simulated conductances into isolated cardiomyocytes in a real-time continuous loop, dynamic clamp has greatly expanded the capabilities of patch clamp outside traditional static voltage and current protocols. Recent applications include fine manipulation of injected artificial conductances to identify promising drug targets in the prevention of arrhythmia and the direct testing of model-based hypotheses. Furthermore, dynamic clamp has been used to enhance existing experimental models by addressing their intrinsic limitations, which increased predictive power in identifying pro-arrhythmic pharmacological compounds. Here, we review the recent advances of the dynamic clamp technique in cardiac electrophysiology with a focus on its future role in the development of safety testing and discovery of anti-arrhythmic drugs.

Dynamics of excitable cells and networks depend on the membrane time constant, set by membrane resistance and capacitance. Whereas pharmacological and genetic manipulations of ionic conductances of excitable membranes are routine in electrophysiology, experimental control over capacitance remains a challenge. Here, we present capacitance clamp, an approach that allows electrophysiologists to mimic a modified capacitance in biological neurons via an unconventional application of the dynamic clamp technique. We first demonstrate the feasibility to quantitatively modulate capacitance in a mathematical neuron model and then confirm the functionality of capacitance clamp in in vitro experiments in granule cells of rodent dentate gyrus with up to threefold virtual capacitance changes. Clamping of capacitance thus constitutes a novel technique to probe and decipher mechanisms of neuronal signaling in ways that were so far inaccessible to experimental electrophysiology.

This work introduces the first atrial-specific in-silico human induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs) model, based on a set of phenotype-specific I Kur ,I KCa and I K1 membrane currents. This model is built on novel in-vitro experimental data recently published by some of the co-authors to simulate the paced action potential of matured atrial-like hiPSC-CMs. The model consists of a system of stiff ordinary differential equations depending on several parameters, which have been tuned by automatic optimization techniques to closely match selected experimental biomarkers. The new model effectively simulates the electronic in-vitro hiPSC-CMs maturation process, transitioning from an unstable depolarized membrane diastolic potential to a stable hyperpolarized resting potential, and exhibits spontaneous firing activity in unpaced conditions. Moreover, our model accurately reflects the experimental rate dependence data at different cycle length and demonstrates the expected response to a specific current blocker. This atrial-specific in-silico model provides a novel computational tool for electrophysiological studies of cardiac stem cells and their applications to drug evaluation and atrial fibrillation treatment., Competing Interests: Declaration of competing interest There are no conflicts of interest: None Declared., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Reciprocal inhibition is a building block in many sensory and motor circuits. We studied the features that underly robustness in reciprocally inhibitory two neuron circuits. We used the dynamic clamp to create reciprocally inhibitory circuits from pharmacologically isolated neurons of the crab stomatogastric ganglion by injecting artificial graded synaptic (ISyn) and hyperpolarization-activated inward (IH) currents. There is a continuum of mechanisms in circuits that generate antiphase oscillations, with ‘release’ and ‘escape’ mechanisms at the extremes, and mixed mode oscillations between these extremes. In release, the active neuron primarily controls the off/on transitions. In escape, the inhibited neuron controls the transitions. We characterized the robustness of escape and release circuits to alterations in circuit parameters, temperature, and neuromodulation. We found that escape circuits rely on tight correlations between synaptic and H conductances to generate bursting but are resilient to temperature increase. Release circuits are robust to variations in synaptic and H conductances but fragile to temperature increase. The modulatory current (IMI) restores oscillations in release circuits but has little effect in escape circuits. Perturbations can alter the balance of escape and release mechanisms and can create mixed mode oscillations. We conclude that the same perturbation can have dramatically different effects depending on the circuits’ mechanism of operation that may not be observable from basal circuit activity.

The congenital short QT syndrome (SQTS) is a cardiac condition that leads to abbreviated ventricular repolarization and an increased susceptibility to arrhythmia and sudden death. The SQT3 form of the syndrome is due to mutations to the KCNJ2 gene that encodes Kir2.1, a critical component of channels underlying cardiac inwardly rectifying K+ current, IK1. The first reported SQT3 KCNJ2 mutation gives rise to the D172N Kir2.1 mutation, the consequences of which have been studied on recombinant channels in vitro and in ventricular cell and tissue simulations. The aim of this study was to establish the effects of the D172N mutation on ventricular repolarization through real-time replacement of IK1 using the dynamic clamp technique. Whole-cell patch-clamp recordings were made from adult guinea-pig left ventricular myocytes at physiological temperature. Action potentials (APs) were elicited at 1 Hz. Intrinsic IK1 was inhibited with a low concentration (50 µM) of Ba2+ ions, which led to AP prolongation and triangulation, accompanied by a ∼6 mV depolarization of resting membrane potential. Application of synthetic IK1 through dynamic clamp restored AP duration, shape and resting potential. Replacement of wild-type (WT) IK1 with heterozygotic (WT-D172N) or homozygotic (D172N) mutant formulations under dynamic clamp significantly abbreviated AP duration (APD90) and accelerated maximal AP repolarization velocity, with no significant hyperpolarization of resting potential. Across stimulation frequencies from 0.5 to 3 Hz, the relationship between APD90 and cycle length was downward shifted, reflecting AP abbreviation at all stimulation frequencies tested. In further AP measurements at 1 Hz from hiPSC cardiomyocytes, the D172N mutation produced similar effects on APD and repolarization velocity; however, resting potential was moderately hyperpolarized by application of mutant IK1 to these cells. Overall, the results of this study support the major changes in ventricular cell AP repolarization with the D172N predicted from prior AP modelling and highlight the potential utility of using adult ventricular cardiomyocytes for dynamic clamp exploration of functional consequences of Kir2.1 mutations.

The congenital short QT syndrome (SQTS) is a cardiac condition that leads to abbreviated ventricular repolarization and an increased susceptibility to arrhythmia and sudden death. The SQT3 form of the syndrome is due to mutations to the KCNJ2 gene that encodes Kir2.1, a critical component of channels underlying cardiac inwardly rectifying K+ current, IK1. The first reported SQT3 KCNJ2 mutation gives rise to the D172N Kir2.1 mutation, the consequences of which have been studied on recombinant channels in vitro and in ventricular cell and tissue simulations. The aim of this study was to establish the effects of the D172N mutation on ventricular repolarization through real-time replacement of IK1 using the dynamic clamp technique. Whole-cell patch-clamp recordings were made from adult guinea-pig left ventricular myocytes at physiological temperature. Action potentials (APs) were elicited at 1 Hz. Intrinsic IK1 was inhibited with a low concentration (50 µM) of Ba2+ ions, which led to AP prolongation and triangulation, accompanied by a ∼6 mV depolarization of resting membrane potential. Application of synthetic IK1 through dynamic clamp restored AP duration, shape and resting potential. Replacement of wild-type (WT) IK1 with heterozygotic (WT-D172N) or homozygotic (D172N) mutant formulations under dynamic clamp significantly abbreviated AP duration (APD90) and accelerated maximal AP repolarization velocity, with no significant hyperpolarization of resting potential. Across stimulation frequencies from 0.5 to 3 Hz, the relationship between APD90 and cycle length was downward shifted, reflecting AP abbreviation at all stimulation frequencies tested. In further AP measurements at 1 Hz from hiPSC cardiomyocytes, the D172N mutation produced similar effects on APD and repolarization velocity; however, resting potential was moderately hyperpolarized by application of mutant IK1 to these cells. Overall, the results of this study support the major changes in ventricular cell AP repolarization with the D172N predicted from prior AP modelling and highlight the potential utility of using adult ventricular cardiomyocytes for dynamic clamp exploration of functional consequences of Kir2.1 mutations. [ABSTRACT FROM AUTHOR]

BackgroundTransient outward K currents (Ito) have been reported both to suppress and to facilitate early afterdepolarizations (EADs) when repolarization reserve is reduced. Here, we used the dynamic clamp technique to analyze how Ito accounts for these paradoxical effects on EADs by influencing the dynamic evolution of repolarization reserve during the action potential.Methods and resultsIsolated patch-clamped rabbit ventricular myocytes were exposed to either oxidative stress (H2O2) or hypokalemia to induce bradycardia-dependent EADs at a long pacing cycle length of 6 s, when native rabbit Ito is substantial. EADs disappeared when the pacing cycle length was shortened to 1 s, when Ito becomes negligible because of incomplete recovery from inactivation. During 6-s pacing cycle length, EADs were blocked by the Ito blocker 4-aminopyridine, but reappeared when a virtual current with appropriate Ito-like properties was reintroduced using the dynamic clamp (n=141 trials). During 1-s pacing cycle length in the absence of 4-aminopyridine, adding a virtual Ito-like current (n=1113 trials) caused EADs to reappear over a wide range of Ito conductance (0.005-0.15 nS/pF), particularly when inactivation kinetics were slow (τinact≥20 ms) and the pedestal (noninactivating component) was small (

Modulators are generally expected to establish a network configuration that is appropriate for the current circumstances. We characterize a situation where the opposite is apparently observed. A network effect of a peptide modulator is counterproductive in that it tends to impede rather than promote the creation of the configuration that is appropriate when the modulator is released. This raises a question: why does release occur? We present data that strongly suggest that it impacts task switching. Our experiments were conducted in an Aplysia feeding network that generates egestive and ingestive motor programs. Initial experiments focused on egestive activity and the neuron B8. As activity becomes egestive, there is an increase in synaptic drive to B8 and its firing frequency increases (Wang et al., 2019). We show that, as this occurs, there is also a persistent current that develops in B8 that is outward rather than inward. Dynamic clamp introduction of this current decreases excitability. When there is an egestive-ingestive task switch in Aplysia, negative biasing is observed (i.e., a bout of egestive activity has a negative impact on a subsequent attempt to initiate an ingestive response) (Proekt et al., 2004). Using an in vitro analog of negative biasing, we demonstrate that the outward current that develops during egestive priming plays an important role in establishing this phenomenon. Our data suggest that, although the outward current induced as activity becomes egestive is counterproductive at the time, it plays an anticipatory role in that it subsequently impacts task switching. [ABSTRACT FROM AUTHOR]

Atrial fibrillation (AF) is the most common cardiac arrhythmia. About 5–15% of AF patients have a mutation in a cardiac gene, including mutations in KCNA5, encoding the Kv1.5 α-subunit of the ion channel carrying the atrial-specific ultrarapid delayed rectifier K+ current (IKur). Both loss-of-function and gain-of-function AF-related mutations in KCNA5 are known, but their effects on action potentials (APs) of human cardiomyocytes have been poorly studied. Here, we assessed the effects of wild-type and mutant IKur on APs of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). We found that atrial-like hiPSC-CMs, generated by a retinoic acid-based differentiation protocol, have APs with faster repolarization compared to ventricular-like hiPSC-CMs, resulting in shorter APs with a lower AP plateau. Native IKur, measured as current sensitive to 50 μM 4-aminopyridine, was 1.88 ± 0.49 (mean ± SEM, n = 17) and 0.26 ± 0.26 pA/pF (n = 17) in atrial- and ventricular-like hiPSC-CMs, respectively. In both atrial- and ventricular-like hiPSC-CMs, IKur blockade had minimal effects on AP parameters. Next, we used dynamic clamp to inject various amounts of a virtual IKur, with characteristics as in freshly isolated human atrial myocytes, into 11 atrial-like and 10 ventricular-like hiPSC-CMs, in which native IKur was blocked. Injection of IKur with 100% density shortened the APs, with its effect being strongest on the AP duration at 20% repolarization (APD20) of atrial-like hiPSC-CMs. At IKur densities < 100% (compared to 100%), simulating loss-of-function mutations, significant AP prolongation and raise of plateau were observed. At IKur densities > 100%, simulating gain-of-function mutations, APD20 was decreased in both atrial- and ventricular-like hiPSC-CMs, but only upon a strong increase in IKur. In ventricular-like hiPSC-CMs, lowering of the plateau resulted in AP shortening. We conclude that a decrease in IKur, mimicking loss-of-function mutations, has a stronger effect on the AP of hiPSC-CMs than an increase, mimicking gain-of-function mutations, whereas in ventricular-like hiPSC-CMs such increase results in AP shortening, causing their AP morphology to become more atrial-like. Effects of native IKur modulation on atrial-like hiPSC-CMs are less pronounced than effects of virtual IKur injection because IKur density of atrial-like hiPSC-CMs is substantially smaller than that of freshly isolated human atrial myocytes.

Neural competition plays an essential role in active selection processes of noisy and ambiguous input signals, and it is assumed to underlie emergent properties of brain functioning, such as perceptual organization and decision-making. Despite ample theoretical research on neural competition, experimental tools to allow neurophysiological investigation of competing neurons have not been available. We developed a "hybrid" system where real-life neurons and a computer-simulated neural circuit interacted. It enabled us to construct a mutual inhibition circuit between two real-life pyramidal neurons. We then asked what dynamics this minimal unit of neural competition exhibits and compared them with the known behavioral-level dynamics of neural competition. We found that the pair of neurons shows bistability when activated simultaneously by current injections. The addition of modeled synaptic noise and changes in the activation strength showed that the dynamics of the circuit are strikingly similar to the known properties of bistable visual perception: The distribution of dominance durations showed a right-skewed shape, and the changes of the activation strengths caused changes in dominance, dominance durations, and reversal rates as stated in the well-known empirical laws of bistable perception known as Levelt's propositions. [ABSTRACT FROM AUTHOR]

The rostral nucleus of the solitary tract (rNST) serves as the first central relay in the gustatory system. In addition to synaptic interactions, central processing is also influenced by the ion channel composition of individual neurons. For example, voltage-gated K+ channels such as outward K+ current (IA) can modify the integrative properties of neurons. IA currents are prevalent in rNST projection cells but are also found to a lesser extent in GABAergic interneurons. However, characterization of the kinetic properties of IA, the molecular basis of these currents, as well as the consequences of IA on spiking properties of identified rNST cells is lacking. Here, we show that IA in rNST GABAergic (G+) and non-GABAergic (G-) neurons share a common molecular basis. In both cell types, there was a reduction in IA following treatment with the specific Kv4 channel blocker AmmTx3. However, the kinetics of activation and inactivation of IA in the two cell types were different with G- neurons having significantly more negative half-maximal activation and inactivation values. Likewise, under current clamp, G- cells had significantly longer delays to spike initiation in response to a depolarizing stimulus preceded by a hyperpolarizing prepulse. Computational modeling and dynamic clamp suggest that differences in the activation half-maximum may account for the differences in delay. We further observed evidence for a window current under both voltage clamp and current clamp protocols. We speculate that the location of Kv4.3 channels on dendrites, together with a window current for IA at rest, serves to regulate excitatory afferent inputs. NEW & NOTEWORTHY Here, we demonstrate that the transient outward K+ current IA occurs in both GABAergic and nonGABAergic neurons via Kv4.3 channels in the rostral (gustatory) solitary nucleus. Although found in both cell types, IA is more prevalent in non-GABAergic cells; a larger conductance at more negative potentials leads to a greater impact on spike initiation compared with GABAergic neurons. An IA window current further suggests that IA can regulate excitatory afferent input to the nucleus. [ABSTRACT FROM AUTHOR]

Stellate cells (SCs) of the medial entorhinal cortex (MEC) are rich in hyperpolarization‐activated cyclic nucleotide–gated (HCN) channels, which are known to effectively shape their activity patterns. The explanatory mechanisms, however, have remained elusive. One important but previously unassessed possibility is that HCN channels control the gain of synaptic inputs to these cells. Here, we test this possibility in rat brain slices, while subjecting SCs to a stochastic synaptic bombardment using the dynamic clamp. We show that in the presence of synaptic noise, HCN channels mainly exert their influence by increasing the relative signal gain in the theta frequency through the theta modulation of stochastic synaptic inputs. This subthreshold synaptic modulation then translates into a spiking resonance, which steepens with excitation in the presence of HCN channels. We present here a systematic assessment of synaptic theta modulation and trace its implications to the suprathreshold control of firing rate motifs. Such analysis was yet lacking in the SC literature. Furthermore, we assess the impact of noise statistics on this gain modulation and indicate possible mechanisms for the emergence of membrane theta oscillations and synaptic ramps, as observed in vivo. We support the data with a computational model that further unveils a competing role of inhibition, suggesting important implications for MEC computations. [ABSTRACT FROM AUTHOR]

Hybrid circuits built by creating mono- or bi-directional interactions among living cells and model neurons and synapses are an effective way to study neuron, synaptic and neural network dynamics. However, hybrid circuit technology has been largely underused in the context of neuroscience studies mainly because of the inherent difficulty in implementing and tuning this type of interactions. In this paper, we present a set of algorithms for the automatic adaptation of model neurons and connections in the creation of hybrid circuits with living neural networks. The algorithms perform model time and amplitude scaling, real-time drift adaptation, goal-driven synaptic and model tuning/calibration and also automatic parameter mapping. These algorithms have been implemented in RTHybrid, an open-source library that works with hard real-time constraints. We provide validation examples by building hybrid circuits in a central pattern generator. The results of the validation experiments show that the proposed dynamic adaptation facilitates closed-loop communication among living and artificial model neurons and connections, and contributes to characterize system dynamics, achieve control, automate experimental protocols and extend the lifespan of the preparations. [ABSTRACT FROM AUTHOR]

We previously demonstrated that a two-cell syncytium, composed of a ventricular myocyte and an mHCN2 expressing cell, recapitulated most properties of in vivo biological pacing induced by mHCN2-transfected hMSCs in the canine ventricle. Here, we use the two-cell syncytium, employing dynamic clamp, to study the roles of gf (pacemaker conductance), gK1 (background K+ conductance), and gj (intercellular coupling conductance) in biological pacing. We studied gf and gK1 in single HEK293 cells expressing cardiac sodium current channel Nav1.5 (SCN5A). At fixed gf, increasing gK1 hyperpolarized the cell and initiated pacing. As gK1 increased, rate increased, then decreased, finally ceasing at membrane potentials near EK. At fixed gK1, increasing gf depolarized the cell and initiated pacing. With increasing gf, rate increased reaching a plateau, then decreased, ceasing at a depolarized membrane potential. We studied gj via virtual coupling with two non-adjacent cells, a driver (HEK293 cell) in which gK1 and gf were injected without SCN5A and a follower (HEK293 cell), expressing SCN5A. At the chosen values of gK1 and gf oscillations initiated in the driver, when gj was increased synchronized pacing began, which then decreased by about 35% as gj approached 20 nS. Virtual uncoupling yielded similar insights into gj. We also studied subthreshold oscillations in physically and virtually coupled cells. When coupling was insufficient to induce pacing, passive spread of the oscillations occurred in the follower. These results show a non-monotonic relationship between gK1, gf, gj, and pacing. Further, oscillations can be generated by gK1 and gf in the absence of SCN5A. [ABSTRACT FROM AUTHOR]

Across biological systems, cooperativity between proteins enables fast actions, supra-linear responses, and long-lasting molecular switches. In the nervous system, however, the function of cooperative interactions between voltage-dependent ionic channels remains largely unknown. Based on mathematical modeling, we here demonstrate that clusters of strongly cooperative ion channels can plausibly form bistable conductances. Consequently, clusters are permanently switched on by neuronal spiking, switched off by strong hyperpolarization, and remain in their state for seconds after stimulation. The resulting short-term memory of the membrane potential allows to generate persistent firing when clusters of cooperative channels are present together with non-cooperative spike-generating conductances. Dynamic clamp experiments in rodent cortical neurons confirm that channel cooperativity can robustly induce graded persistent activity – a single-cell based, multistable mnemonic firing mode experimentally observed in several brain regions. We therefore propose that ion channel cooperativity constitutes an efficient cell-intrinsic implementation for short-term memories at the voltage level.

Voltage-gated sodium channels play a critical role in cellular excitability, amplifying small membrane depolarizations into action potentials. Interactions with auxiliary subunits and other factors modify the intrinsic kinetic mechanism to result in new molecular and cellular functionality. We show here that sodium channels can implement a molecular leaky integrator, where the input signal is the membrane potential and the output is the occupancy of a long-term inactivated state. Through this mechanism, sodium channels effectively measure the frequency of action potentials and convert it into Na+ current availability. In turn, the Na+ current can control neuronal firing frequency in a negative feedback loop. Consequently, neurons become less sensitive to changes in excitatory input and maintain a lower firing rate. We present these ideas in the context of rat serotonergic raphe neurons, which fire spontaneously at low frequency and provide critical neuromodulation to many autonomous and cognitive brain functions.

Excitability--a threshold-governed transient in transmembrane voltage--is a fundamental physiological process that controls the function of the heart, endocrine, muscles, and neuronal tissues. The 1950s Hodgkin and Huxley explicit formulation provides a mathematical framework for understanding excitability, as the consequence of the properties of voltage-gated sodium and potassium channels. The Hodgkin-Huxley model is more sensitive to parametric variations of protein densities and kinetics than biological systems whose excitability is apparently more robust. It is generally assumed that the model's sensitivity reflects missing functional relations between its parameters or other components present in biological systems. Here we experimentally assembled excitable membranes using the dynamic clamp and voltage-gated potassium ionic channels (Kv1.3) expressed in Xenopus oocytes. We take advantage of a theoretically derived phase diagram, where the phenomenon of excitability is reduced to two dimensions defined as combinations of the Hodgkin-Huxley model parameters, to examine functional relations in the parameter space. Moreover, we demonstrate activity dependence and hysteretic dynamics over the phase diagram due to the impacts of complex slow inactivation kinetics. The results suggest that maintenance of excitability amid parametric variation is a low-dimensional, physiologically tenable control process. In the context of model construction, the results point to a potentially significant gap between high-dimensional models that capture the full measure of complexity displayed by ion channel function and the lower dimensionality that captures physiological function. [ABSTRACT FROM AUTHOR]

As in mammals, Drosophila circadian clock neurons display rhythms of activity with higher action potential firing rates and more positive resting membrane potentials during the day. This rhythmic excitability has been widely observed but, critically, its regulation remains unresolved. We have characterized and modelled the changes underlying these electrical activity rhythms in the lateral ventral clock neurons (LNvs). We show that currents mediated by the voltage‐gated potassium channels Shaw (Kv3) and Shal (Kv4) oscillate in a circadian manner. Disruption of these channels, by expression of dominant negative (DN) subunits, leads to changes in circadian locomotor activity and shortens lifespan. LNv whole‐cell recordings then show that changes in Shaw and Shal currents drive changes in action potential firing rate and that these rhythms are abolished when the circadian molecular clock is stopped. A whole‐cell biophysical model using Hodgkin‐Huxley equations can recapitulate these changes in electrical activity. Based on this model and by using dynamic clamp to manipulate clock neurons directly, we can rescue the pharmacological block of Shaw and Shal, restore the firing rhythm, and thus demonstrate the critical importance of Shaw and Shal. Together, these findings point to a key role for Shaw and Shal in controlling circadian firing of clock neurons and show that changes in clock neuron currents can account for this. Moreover, with dynamic clamp we can switch the LNvs between morning‐like and evening‐like states of electrical activity. We conclude that changes in Shaw and Shal underlie the daily oscillation in LNv firing rate. [ABSTRACT FROM AUTHOR]

Synaptic and intrinsic properties interact to sculpt neuronal output. Kisspeptin neurons in the hypothalamic arcuate nucleus help convey homeostatic estradiol feedback to central systems controlling fertility. Estradiol increases membrane depolarization induced by GABAA receptor activation in these neurons. We hypothesized that the mechanisms underlying estradiol-induced alterations in postsynaptic response to GABA, and also AMPA, receptor activation include regulation of voltage-gated potassium currents. Whole-cell recordings of arcuate kisspeptin neurons in brain slices from ovariectomized (OVX) and OVX+estradiol (OVX+E) female mice during estradiol negative feedback revealed that estradiol reduced capacitance, reduced transient and sustained potassium currents, and altered voltage dependence and kinetics of transient currents. Consistent with these observations, estradiol reduced rheobase and action potential latency. To study more directly interactions between synaptic and active intrinsic estradiol feedback targets, dynamic clamp was used to simulate GABA and AMPA conductances. Both GABA and AMPA dynamic clamp-induced postsynaptic potentials (PSPs) were smaller in neurons from OVX than OVX+E mice; blocking transient potassium currents eliminated this difference. To interrogate the role of the estradiol-induced changes in passive intrinsic properties, different Markov model structures based on the properties of the transient potassium current in cells from OVX or OVX+E mice were combined in silico with passive properties reflecting these two endocrine conditions. Some of tested models reproduced the effect on PSPs in silico, revealing that AMPA PSPs were more sensitive to changes in capacitance. These observations support the hypothesis that PSPs in arcuate kisspeptin neurons are regulated by estradiol-sensitive mechanisms including potassium conductances and membrane properties. [ABSTRACT FROM AUTHOR]

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease in which motor neurons degenerate, resulting in muscle atrophy, paralysis, and fatality. Studies using mouse models of ALS indicate a protracted period of disease development with progressive motor neuron pathology, evident as early as embryonic and postnatal stages. Key missing information includes concomitant alterations in the sensorimotor circuit essential for normal development and function of the neuromuscular system. Leveraging unique brainstem circuitry, we show in vitro evidence for reflex circuit-specific postnatal abnormalities in the jaw proprioceptive sensory neurons in the well-studied SOD1G93A mouse. These include impaired and arrhythmic action potential burst discharge associated with a deficit in Nav1.6 Na+ channels. However, the mechanoreceptive and nociceptive trigeminal ganglion neurons and the visual sensory retinal ganglion neurons were resistant to excitability changes in age-matched SOD1G93A mice. Computational modeling of the observed disruption in sensory patterns predicted asynchronous self-sustained motor neuron discharge suggestive of imminent reflexive defects, such as muscle fasciculations in ALS. These results demonstrate a novel reflex circuit-specific proprioceptive sensory abnormality in ALS. [ABSTRACT FROM AUTHOR]

Correlated electrical activity in neurons is a prominent characteristic of cortical microcircuits. Despite a growing amount of evidence concerning both spike-count and subthreshold membrane potential pairwise correlations, little is known about how different types of cortical neurons convert correlated inputs into correlated outputs. We studied pyramidal neurons and two classes of GABAergic interneurons of layer 5 in neocortical brain slices obtained from rats of both sexes, and we stimulated them with biophysically realistic correlated inputs, generated using dynamic clamp. We found that the physiological differences between cell types manifested unique features in their capacity to transfer correlated inputs. We used linear response theory and computational modeling to gain clear insights into how cellular properties determine both the gain and timescale of correlation transfer, thus tying single-cell features with network interactions. Our results provide further ground for the functionally distinct roles played by various types of neuronal cells in the cortical microcircuit. [ABSTRACT FROM AUTHOR]

For a biological neural network to be functional, its neurons need to be connected with synapses of appropriate strength, and each neuron needs to appropriately respond to its synaptic inputs. This second aspect of network tuning is maintained by intrinsic plasticity; yet it is often considered secondary to changes in connectivity and mostly limited to adjustments of overall excitability of each neuron. Here we argue that even nonoscillatory neurons can be tuned to inputs of different temporal dynamics and that they can routinely adjust this tuning to match the statistics of their synaptic activation. Using the dynamic clamp technique, we show that, in the tectum of Xenopus tadpole, neurons become selective for faster inputs when animals are exposed to fast visual stimuli but remain responsive to longer inputs in animals exposed to slower, looming, or multisensory stimulation. We also report a homeostatic cotuning between synaptic and intrinsic temporal properties of individual tectal cells. These results expand our understanding of intrinsic plasticity in the brain and suggest that there may exist an additional dimension of network tuning that has been so far overlooked. NEW & NOTEWORTHY We use dynamic clamp to show that individual neurons in the tectum of Xenopus tadpoles are selectively tuned to either shorter (more synchronous) or longer (less synchronous) synaptic inputs. We also demonstrate that this intrinsic temporal tuning is strongly shaped by sensory experiences. This new phenomenon, which is likely to be mediated by changes in sodium channel inactivation, is bound to have important consequences for signal processing and the development of local recurrent connections. [ABSTRACT FROM AUTHOR]

In motor systems, higher-order neurons provide commands to lower-level central pattern generators (CPGs) that autonomously produce rhythmic motor patterns. Such hierarchical organization is often thought to be inherent in the anatomical position of the neurons. Here, however, we report that a neuron that is member of a CPG in one species acts as a higher-order neuron in another species. In the nudibranch mollusc, Melibe leonina, swim interneuron 1 (Sil) is in the CPG underlying swimming, firing rhythmic bursts of action potentials as part of the swim motor pattern. We found that its homolog in another nudibranch, Dendronotus iris, serves as a neuromodulatory command neuron for the CPG of a homologous swimming behavior. In Dendronotus, Sil fired irregularly throughout the swim motor pattern. The burst and spike frequencies of Dendronotus swim CPG neurons correlated with Si 1 firing frequency. Si 1 activity was both necessary and sufficient for the initiation and maintenance of the swim motor pattern. Each Si 1 was electrically coupled to all of the CPG neurons and made monosynaptic excitatory synapses with both Si3s. Sil also bilaterally potentiated the excitatory synapse from Si3 to Si2. "Virtual neuromodulation" of both Si3-to-Si2 synapses using dynamic clamp combined with depolarization of both Si3s mimicked the effects of Sil stimulation on the swim motor pattern. Thus, in Dendronotus, Sil is a command neuron that turns on, maintains, and accelerates the motor pattern through synaptic and neuromodulatory actions, thereby differing from its homolog in Melibe in its functional position in the motor hierarchy. [ABSTRACT FROM AUTHOR]

Central output of gonadotropin-releasing hormone (GnRH) neurons controls fertility and is sculpted by sex-steroid feedback. A switch of estradiol action from negative to positive feedback initiates a surge of GnRH release, culminating in ovulation. In ovariectomized mice bearing constant-release estradiol implants (OVX+E), GnRH neuron firing is suppressed in the morning (AM) by negative feedback and activated in the afternoon (PM) by positive feedback; no time-of-day-dependent changes occur in OVX mice. In this daily surge model, GnRH neuron intrinsic properties are shifted to favor increased firing during positive feedback. It is unclear whether this shift and the observed concomitant increase in GABAergic transmission, which typically excites GnRH neurons, are independently sufficient for increasing GnRH neuron firing rate during positive feedback or whether both are needed. To test this, we used dynamic clamp to inject selected previously recorded trains of GABAergic postsynaptic conductances (PSgs) collected during the different feedback states of the daily surge model into GnRH neurons from OVX, OVX+E AM, and OVX+E PM mice. PSg trains mimicking positive feedback initiated more action potentials in cells from OVX+E PM mice than negative feedback or OVX (open feedback loop) trains in all three animal models, but the positive-feedback train was most effective when applied to cells during positive feedback. In silico studies of model GnRH neurons in which >1000 PSg trains were tested exhibited the same results. These observations support the hypothesis that GnRH neurons integrate fast-synaptic and intrinsic changes to increase firing rates during positive feedback. [ABSTRACT FROM AUTHOR]

Sensory systems exploit parallel processing of stimulus features to enable rapid, simultaneous extraction of information. Mechanisms that facilitate this differential extraction of stimulus features can be intrinsic or synaptic in origin. A subdivision of the avian cochlear nucleus, nucleus angularis (NA), extracts sound intensity information from the auditory nerve and contains neurons that exhibit diverse responses to sound and current injection. NA neurons project to multiple regions ascending the auditory brain stem including the superior olivary nucleus, lateral lemniscus, and avian inferior colliculus, with functional implications for inhibitory gain control and sound localization. Here we investigated whether the diversity of auditory response patterns in NA can be accounted for by variation in intrinsic physiological features. Modeled sound-evoked auditory nerve input was applied to NA neurons with dynamic clamp during in vitro whole cell recording at room temperature. Temporal responses to auditory nerve input depended on variation in intrinsic properties, and the low-threshold K+ current was implicated as a major contributor to temporal response diversity and neuronal inputoutput functions. An auditory nerve model of acoustic amplitude modulation produced synchrony coding of modulation frequency that depended on the intrinsic physiology of the individual neuron. In Primary-Like neurons, varying low-threshold K+ conductance with dynamic clamp altered temporal modulation tuning bidirectionally. Taken together, these data suggest that intrinsic physiological properties play a key role in shaping auditory response diversity to both simple and more naturalistic auditory stimuli in the avian cochlear nucleus. NEW & NOTEWORTHY This article addresses the question of how the nervous system extracts different information in sounds. Neurons in the cochlear nucleus show diverse responses to acoustic stimuli that may allow for parallel processing of acoustic features. The present studies suggest that diversity in intrinsic physiological features of individual neurons, including levels of a low voltage-activated K current, play a major role in regulating the diversity of auditory responses. [ABSTRACT FROM AUTHOR]

The dynamic clamp technique has emerged as a powerful tool in the field of cardiac electrophysiology, enabling researchers to investigate the intricate dynamics of ion currents in cardiac cells. Potassium channels play a critical role in the functioning of cardiac cells and the overall electrical stability of the heart. This chapter provides a comprehensive overview of the methods and applications of dynamic clamp in the study of key potassium currents in cardiac cells. A step-by-step guide is presented, detailing the experimental setup and protocols required for implementing the dynamic clamp technique in cardiac cell studies. Special attention is given to the design and construction of a dynamic clamp setup with Real Time eXperimental Interface, configurations, and the incorporation of mathematical models to mimic ion channel behavior. The chapter's core focuses on applying dynamic clamp to elucidate the properties of various potassium channels in cardiac cells. It discusses how dynamic clamp can be used to investigate channel kinetics, voltage-dependent properties, and the impact of different potassium channel subtypes on cardiac electrophysiology. The chapter will also include examples of specific dynamic clamp experiments that studied potassium currents or their applications in cardiac cells., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The classic dynamic clamp technique uses a real-time electrical interface between living cells and neural simulations in order to investigate hypotheses about neural function and structure. One of the acknowledged drawbacks of that technique is the limited control of the cells’ chemical microenvironment. In this manuscript, we use a novel combination of nanosensor and microfluidic technology and microfluidic and neural simulations to add sensing and control of chemical concentrations to the dynamic clamp technique. Specifically, we use a microfluidic lab-on-a-chip to generate distinct chemical concentration gradients (ions or neuromodulators), to register the concentrations with embedded nanosensors and use the processed signals as an input to simulations of a neural cell. The ultimate goal of this project is to close the loop and provide sensor signals to the microfluidic lab-on-a-chip to mimic the interaction of the simulated cell with other cells in its chemical environment.

Pain is a complex process that involves both detection in the peripheral nervous system and perception in the CNS. Individual-to-individual differences in pain are well documented, but not well understood. Here we capitalized on inherited erythromelalgia (IEM), a well characterized human genetic model of chronic pain, and studied a unique family containing related IEM subjects with the same disease-causing NaV1.7 mutation, which is known to make dorsal root ganglion (DRG) neurons hyperexcitable, but different pain profiles (affected son with severe pain, affected mother with moderate pain, and an unaffected father). We show, first, that, at least in some cases, relative sensitivity to pain can be modeled in subject-specific induced pluripotent stem cell (iPSC)-derived sensory neurons in vitro; second, that, in some cases, mechanisms operating in peripheral sensory neurons contribute to interindividual differences in pain; and third, using whole exome sequencing (WES) and dynamic clamp, we show that it is possible to pinpoint a specific variant of another gene, KCNQ in this particular kindred, that modulates the excitability of iPSC-derived sensory neurons in this family. While different gene variants may modulate DRG neuron excitability and thereby contribute to interindividual differences in pain in other families, this study shows that subject-specific iPSCs can be used to model interindividual differences in pain. We further provide proof-of-principle that iPSCs, WES, and dynamic clamp can be used to investigate peripheral mechanisms and pinpoint specific gene variants that modulate pain signaling and contribute to interindividual differences in pain. [ABSTRACT FROM AUTHOR]

Aim: Repolarization response to β-adrenergic (β-AR) stimulation differs between guinea-pig and canine myocytes and, within the latter, between myocardial layers. Correlative analysis suggests that this may be due to differences in action potential (AP) contour. Here we tested whether AP contour may set the response of current and of repolarization to β-AR stimulation (10 nM isoproterenol, ISO).Methods and results: The responses of AP and current to ISO were measured under I-clamp and "AP-clamp" in guinea-pig (GP), dog epicardial (DEPI) and dog subendocardial (DENDO) myocytes. Dynamic-clamp (DC) was used to evaluate the impact of AP features on AP response to ISO. ISO prolonged AP duration (APD) in GP myocytes, did not affect it in DENDO and shortened it in DEPI ones. The current induced by ISO (IISO) sharply differed between GP and canine myocytes and, to a lesser extent, between DENDO and DEPI ones. Differences in IISO profile likely important in setting APD response (time-to-peak, time-to-reversal), were minimized when canine myocytes where clamped with GP AP-waveforms and vice versa. Introduction of a "notch" in GP AP (by DC) was alone insufficient to affect the APD response to ISO; nevertheless, when incorporated in a GP AP-waveform, the main "canine" AP features ("notch" and low plateau potential) caused IISO of GP myocytes to acquire canine features.Conclusion: Early repolarization contour and level of plateau potential contribute to species-specificity of IISO profile. Changes in AP contour, also when generated by modulation of ISO-insensitive currents, may be crucial in setting APD response to β-AR stimulation. [ABSTRACT FROM AUTHOR]

Key points: Primary somatosensory neurons normally respond to somatic depolarization with transient spiking but can switch to repetitive spiking under pathological conditions. This switch in spiking pattern reflects a qualitative change in spike initiation dynamics and contributes to the hyperexcitability associated with chronic pain. Neurons can be converted to repetitive spiking by adding a virtual conductance using dynamic clamp. By titrating the conductance to determine how much must be added to cause repetitive spiking, we found that small cells are more susceptible to switching (i.e. required less added conductance) than medium–large cells. By measuring how much less conductance is required to cause repetitive spiking when dynamic clamp was combined with other pathomimetic manipulations (e.g. application of inflammatory mediators), we measured how much each manipulation facilitated repetitive spiking. Our results suggest that many pathological factors facilitate repetitive spiking but that the switch to repetitive spiking requires the cumulative effect of many co‐occurring factors. Abstract: Primary somatosensory neurons become hyperexcitable in many chronic pain conditions. Hyperexcitability can include a switch from transient to repetitive spiking during sustained somatic depolarization. This switch results from diverse pathological processes that impact ion channel expression or function. Because multiple pathological processes co‐occur, isolating how much each contributes to switching the spiking pattern is difficult. Our approach to this challenge involves adding a virtual sodium conductance via dynamic clamp. The magnitude of that conductance was titrated to determine the minimum required to enable rheobasic stimulation to evoke repetitive spiking. The minimum required conductance, termed g ¯ Na ∗, was re‐measured before and during manipulations designed to model various pathological processes in vitro. The reduction in g ¯ Na ∗ caused by each pathomimetic manipulation reflects how much the modelled process contributes to switching the spiking pattern. We found that elevating extracellular potassium or applying inflammatory mediators reduced g ¯ Na ∗ whereas direct hyperpolarization had no effect. Inflammatory mediators reduced g ¯ Na ∗ more in medium–large (>30 μm diameter) neurons than in small (⩽30 μm diameter) neurons, but had equivalent effects in cutaneous and muscle afferents. The repetitive spiking induced by dynamic clamp was also found to differ between small and medium–large neurons, thus revealing latent differences in adaptation. Our study demonstrates a novel way to determine to what extent individual pathological factors facilitate repetitive spiking. Our results suggest that most factors facilitate but do not cause repetitive spiking on their own, and, therefore, that a switch to repetitive spiking results from the cumulative effect of many co‐occurring factors. [ABSTRACT FROM AUTHOR]