Conditioning Hyperpolarization Research Articles

Fast A-type currents shape a rapidly adapting form of delayed short latency firing of excitatory superficial dorsal horn neurons that express the neuropeptide Y Y1 receptor.

Neuropeptide Y Y1 receptor-expressing neurons in the dorsal horn of the spinal cord contribute to chronic pain. For the first time, we characterized the firing patterns of Y1-expressing neurons in Y1eGFP reporter mice. Under hyperpolarized conditions, most Y1eGFP neurons exhibited fast A-type potassium currents and delayed, short-latency firing (DSLF). Y1eGFP DSLF neurons were almost always rapidly adapting and often exhibited rebound spiking, characteristics of spinal pain neurons under the control of T-type calcium channels. These results will inspire future studies to determine whether tissue or nerve injury downregulates the channels that underlie A-currents, thus unmasking membrane hyperexcitability in Y1-expressing dorsal horn neurons, leading to persistent pain. Neuroanatomical and behavioural evidence indicates that neuropeptide Y Y1 receptor-expressing interneurons (Y1-INs) in the superficial dorsal horn (SDH) are predominantly excitatory and contribute to chronic pain. Using an adult ex vivo spinal cord slice preparation from Y1eGFP reporter mice, we characterized firing patterns in response to steady state depolarizing current injection of GFP-positive cells in lamina II, the great majority of which expressed Y1 mRNA (88%). Randomly sampled (RS) and Y1eGFP neurons exhibited five firing patterns: tonic, initial burst, phasic, delayed short-latency <180ms (DSLF) and delayed long-latency >180ms (DLLF). When studied at resting membrane potential, most RS neurons exhibited delayed firing, while most Y1eGFP neurons exhibited phasic firing. A preconditioning membrane hyperpolarization produced only subtle changes in the firing patterns of RS neurons, but dramatically shifted Y1eGFP neurons to DSLF (46%) and DLLF (24%). In contrast to RS DSLF neurons, which rarely exhibited spike frequency adaptation, Y1eGFP DSLF neurons were almost always rapidly adapting, a characteristic of nociceptive-responsive SDH neurons. Rebound spiking was more prevalent in Y1eGFP neurons (6% RS vs. 32% Y1eGFP), indicating enrichment of T-type calcium currents. Y1eGFP DSLF neurons exhibited fast A-type potassium currents that are known to delay or limit action potential firing and exhibited smaller current density as compared to RS DSLF neurons. Our results will inspire future studies to determine whether tissue or nerve injury downregulates channels that contribute to A-currents, thus potentially unmasking T-type calcium channel activity and membrane hyperexcitability in Y1-INs, leading to persistent pain.

2077-P: Can Small ß-Cell Subpopulations Control Islet Dynamics? A Theoretical Study

Electrical activity of a pancreatic islet is globally activated under high glucose conditions and globally suppressed under low glucose conditions. This homogeneous behavior occurs due to the highly-coupled nature of the islet, despite heterogeneity of individual β-cells. Using hyperpolarization to silence β-cell activity, studies have shown that small subpopulations of highly functional β-cells can disproportionately control whole islet dynamics. Using a computational model of islet electrophysiology, we studied the theoretical basis of how small highly functional subpopulations of β-cells may regulate the global activity of islets. We modeled metabolic activity of β-cells as a continuous distribution and as a bimodal distribution. Then, in each case, we silenced the highest metabolically active cells and quantified the response of the islet. These highly metabolic cells (GKHigh) were silenced by hyperpolarization, similarly to experimental data. Only under a bimodal distribution, did a small (10%) population of GKHigh cells dramatically reduce islet activity, as experimentally demonstrated. Next, a simulation was run without GKHigh cells present in the islet. When GKHigh cells were absent, the islet remained globally active and had near normal duty cycle, suggesting that these cells are unnecessary for maintaining islet dynamics. Similarly, we investigated another functional subpopulation, β-cells with higher Ca2+ oscillation frequencies which could potentially act as pacemaker cells. When simulating the absence of 10% of these higher frequency cells, the global frequency of the islet was not significantly altered. These results indicate that small highly functional subpopulations are unable to drive islet function under normal conditions, but when targeted under hyperpolarizing conditions may cause dysfunction. Disclosure J.M. Dwulet: None. R.K. Benninger: None. Funding National Institutes of Health (R01DK106412, R01DK102950)

Regulation of intracellular pH by electrogenic Na+/HCO3– co-transporters in embryonic neural stem cell-derived radial glia-like cells

A stroke causes a hypoxic brain microenvironment that alters neural cell metabolism resulting in cell membrane hyperpolarization and intracellular acidosis. We studied how intracellular pH (pHi) is regulated in differentiated mouse neural progenitor cells during hyperpolarizing conditions, induced by prompt reduction of the extracellular K+ concentration. We found that the radial glia-like population in differentiating embryonic neural progenitor cells, but not neuronal cells, was rapidly acidified under these conditions. However, when extracellular calcium was removed, an instant depolarization and recovery of the pHi, back to normal levels, took place. The rapid recovery phase seen in the absence of calcium, was dependent on extracellular bicarbonate and could be inhibited by S0859, a potent Na/HCO3 cotransporter inhibitor. Immunostaining and PCR data, showed that NBCe1 (SLC4A4) and NBCn1 (SLC4A7) were expressed in the cell population and that the pHi recovery in the radial glial-like cells after calcium removal was mediated mainly by the electrogenic sodium bicarbonate transporter NBCe1 (SLC4A4). Our results indicate that extracellular calcium might hamper pHi regulation and Na/HCO3 cotransporter activity in a brain injury microenvironment. Our findings show that the NBC-type transporters are the main pHi regulating systems prevailing in glia-like progenitor cells and that these calcium sensitive transporters are important for neuronal progenitor cell proliferation, survival and neural stem cell differentiation.

Liberalism beyond toleration: Religious exemptions, civility and the ideological other

I address the long-standing problem of toleration in diverse liberal societies in light of the progress of same-sex marriage and continued vehement opposition to it from a significant portion of the population. I advance a view that contrasts with recent discussions by Teresa Bejan, Mere Civility, and especially Cecile Laborde, Liberalism’s Religion. Laborde emphasizes the importance of state sovereignty in fixing the boundaries of church and state, emphasizing the priority of public authority and constitutional supremacy. I argue that emphasis on priority needs to be complemented by a recognition of the importance of forms of reconciliation that go beyond ‘mere civility’. Reflections on toleration in the liberal and democratic traditions – including in the canonical discussions of Locke, Rousseau, Smith and Tocqueville, and in more recent political science – have recognized that the health of liberal democracy benefits enormously from the educative and morally formative resources furnished by religious communities. We must hope and plan for the reconciliation of the values of liberal democracy and the teachings of the major religions in society. In that context, a great source of present difficulty is the political polarization that infects and inflects religious differences. For that reason, I applaud aspects of Justice Kennedy’s opinion in Masterpiece Cakeshop. Kennedy insists on the equal dignity of gay and lesbian couples, but also requires that the complaints of religious wedding vendors should be listened to respectfully. While progressives constantly urge that we do more to include the other – typically meaning refugees, migrants, racial, sexual minorities and so on – one great challenge in conditions of hyper polarization is to include the ideological other.

Partisan Constitutionalism: Reconsidering the Role of Political Parties in Popular Constitutional Change

Scholars have long understood that political parties play an important role in shaping constitutional culture over time. This occurs most obviously through judicial appointments but also by participating in the shaping of public opinion and passing legislation affecting the scope of our fundamental commitments. But recent legal challenges to the healthcare law highlight the ability of political parties to use courts to shift the scope and meaning of our policy and constitutional commitments also in the very short run, through their strategic support for high-stakes public litigation. In both the NFIB and King challenges, some number of political opponents of the healthcare law recognized in the developing legal case an opportunity to extend a fight that had been waged in the political branches (at both the state and federal level), to a new and potentially more favorable battleground. This paper seeks to identify, for the first time, the mechanisms that allow political parties to play this role. I use the healthcare challenges to show that parties, acting under certain conditions, can increase both the success and also speed at which politically-charged legal claims are dislodged from off-the-wall to being fair game for courts. In particular, I argue that this happens through four main mechanisms, each of which can be observed in two healthcare cases. Political parties have a unique ability, beyond what is available to private litigants or social movements, to initiate and support legal challenges by 1) providing the direct infrastructure for public litigation, 2) communicating salience of the challenge to courts, including by signaling the case’s importance, 3) provide a common vocabulary of constitutional meaning that mediate between the litigants, the courts, and the public, and 4) framing relevant prudential considerations that might influence judicial decisionmaking. I describe how these four connected mechanisms were used by the party apparatus — through a process I term “partisan constitutionalism” — to advance the ACA challengers’ legal claims. I show that these dynamics helped move the challengers’ arguments from being widely considered unthinkable to being open and contested questions — thus providing cover for courts to depart from settled precedent to rule in favor of the challengers. This paper seeks to show the different ways that parties can support strategic constitutional litigation — while also acknowledging that various political and institutional considerations will often prevent them from playing this role. I conclude that partisan constitutionalism has been made both more attractive and feasible by the background conditions of hyperpolarization and divided government. Rising polarization increases the incentives for political actors to “constitutionalize” political arguments and then for judicial actors to accommodate these attempts, while divided government increases the frequency of situations in which a party finds it advantageous to pursue its policy objectives through strategic litigation rather than orthodox lawmaking. These two features of our modern politics reinforce each other, helping to explain why the party-driven constitutional litigation observed in the healthcare cases is a relatively recent phenomenon — even as many of the mechanisms identified here have long been available to political parties. This dynamic threatens to fundamentally change the role of the judiciary, which increasingly will tend to function — and thus be treated by political actors — as a new institutional veto point for the political process.

P237 Axonal excitability findings in type 1 diabetes mellitus – Median nerve versus tibial nerve comparison

Objective Length dependent peripheral neuropathy is the most common complication of diabetes mellitus (DM). As no curative treatment for diabetic polyneuropathy (DMP) is available, its prevention and early detection is very important. Axonal excitability is defined as the capability of nodal and paranodal pathological changes in DMP. The aim of present study was to determine alterations in axonal excitability findings in tibial nerve by comparing median nerve axonal excitability findings in Type 1 DMP. Methods The total 284 patients, who were followed up for Type 1 DM according to criteria of American Diabetes Association, were screened in this study. Six DMP patients, 10 random non-polyneuropathy cases and 14 healthy control subjects were examined by threshold tracking method (TROND protokol) from median and tibial nerve. Results Even if there is not prominent difference in median nerve axonal excitability studies in PNP and DM patients there was prominent pathological findings in tibial nerve. The strength–duration time constant was significantly longer in DPN Type 1 patients than in normal patients. Supernormality was significantly smaller in DPN patients. Threshold electrotonus studies showed abnormalities in patients with DPN that smaller threshold changes by long depolarizing and hyperpolarizing conditioning current named as “fanning-in”. Conclusion This study confirms that there was significantly alteration in axonal excitability findings in DMP in tibial nerve. Comparisons median and tibial nerve axonal excitability findings have potential to show the length dependent polyneuropathic pathological status in axonal excitability studies.

Omega Mutations along S4 in Nav1.2 Channels Give Insight into Domain Specific Contribution to Activation and Steady State Inactivation

We previously identified the resting state positions of the voltage sensor S4 for each domain of Nav1.2 by means of omega mutations. We found that a double gap is needed to open the omega pore (narrow part of the gating pore) resulting in detectable omega current, also known as gating pore current. At hyperpolarizing conditions, the resting state of S4 was found for double gap RR1,2QQ in domain I, II and IV and for double gap RR2,3QQ in domain III. In this work we evaluated additional conformational states of the voltage sensor S4 moving through the gating pore by further double gap mutations along S4 (second double gap 2,3QQ and third double gap 3,4QQ). Two electrode voltage clamping on X. laevis oocytes expressing rat brain sodium channels Nav1.2 was used to measure macroscopic ionic current through the alpha pore and, if present, omega current through the omega pore. In DI and DII we detected clear outward omega current for S4 mutants RK3,4QQ at depolarizing conditions. Furthermore, we found that activation of sodium currents was right shifted by about 30 mV towards higher potentials compared to the first and second double gap mutants or wild-type sodium channel. These findings suggest two sequential gating steps of S4 between resting and activated state in both domains DI and DII. In DIII and DIV no clear outward omega current could be detected at depolarized potentials either for the second or for the third double gap mutant. However steady state inactivation was strongly left shifted by about 50 to 100 mV to more hyperpolarized potentials for the second and third double gap mutant in both domains, consistent with involvement in recovery from inactivation and immobilization, respectively.

IH activity is increased in populations of slow versus fast motor axons of the rat.

Much is known about the electrophysiological variation in motoneuron somata across different motor units. However, comparatively less is known about electrophysiological variation in motor axons and how this could impact function or electrodiagnosis in healthy or diseased states. We performed nerve excitability testing on two groups of motor axons in Sprague–Dawley rats that are known to differ significantly in their chronic daily activity patterns and in the relative proportion of motor unit types: one group innervating the soleus (“slow motor axons”) and the other group innervating the tibialis anterior (“fast motor axons”) muscles. We found that slow motor axons have significantly larger accommodation compared to fast motor axons upon application of a 100 ms hyperpolarizing conditioning stimulus that is 40% of axon threshold (Z = 3.24, p = 0.001) or 20% of axon threshold (Z = 2.67, p = 0.008). Slow motor axons had larger accommodation to hyperpolarizing currents in the current-threshold measurement (-80% Z = 3.07, p = 0.002; -90% Z = 2.98, p = 0.003). In addition, we found that slow motor axons have a significantly smaller rheobase than fast motor axons (Z = -1.99, p = 0.047) accompanied by a lower threshold in stimulus-response curves. The results provide evidence that slow motor axons have greater activity of the hyperpolarization-activated inwardly rectifying cation conductance (IH) than fast motor axons. It is possible that this difference between fast and slow axons is caused by an adaptation to their chronic differences in daily activity patterns, and that this adaptation might have a functional effect on the motor unit. Moreover, these findings indicate that slow and fast motor axons may react differently to pathological conditions.

Facilitated Hyperpolarization Signaling in Vascular Smooth Muscle-overexpressing TRIC-A Channels

The TRIC channel subtypes, namely TRIC-A and TRIC-B, are intracellular monovalent cation-specific channels and likely mediate counterion movements to support efficient Ca(2+) release from the sarco/endoplasmic reticulum. Vascular smooth muscle cells (VSMCs) contain both TRIC subtypes and two Ca(2+) release mechanisms; incidental opening of ryanodine receptors (RyRs) generates local Ca(2+) sparks to induce hyperpolarization and relaxation, whereas agonist-induced activation of inositol trisphosphate receptors produces global Ca(2+) transients causing contraction. Tric-a knock-out mice develop hypertension due to insufficient RyR-mediated Ca(2+) sparks in VSMCs. Here we describe transgenic mice overexpressing TRIC-A channels under the control of a smooth muscle cell-specific promoter. The transgenic mice developed congenital hypotension. In Tric-a-overexpressing VSMCs from the transgenic mice, the resting membrane potential decreased because RyR-mediated Ca(2+) sparks were facilitated and cell surface Ca(2+)-dependent K(+) channels were hyperactivated. Under such hyperpolarized conditions, L-type Ca(2+) channels were inactivated, and thus, the resting intracellular Ca(2+) levels were reduced in Tric-a-overexpressing VSMCs. Moreover, Tric-a overexpression impaired inositol trisphosphate-sensitive stores to diminish agonist-induced Ca(2+) signaling in VSMCs. These altered features likely reduced vascular tonus leading to the hypotensive phenotype. Our Tric-a-transgenic mice together with Tric-a knock-out mice indicate that TRIC-A channel density in VSMCs is responsible for controlling basal blood pressure at the whole-animal level.

Characterization of the SubstitutedN-Triazole Oxindole TROX-1, a Small-Molecule, State-Dependent Inhibitor of Cav2 Calcium Channels

Biological, genetic, and clinical evidence provide validation for N-type calcium channels (Ca(V)2.2) as therapeutic targets for chronic pain. A state-dependent Ca(V)2.2 inhibitor may provide an improved therapeutic window over ziconotide, the peptidyl Ca(V)2.2 inhibitor used clinically. Supporting this notion, we recently reported that in preclinical models, the state-dependent Ca(V)2 inhibitor (3R)-5-(3-chloro-4-fluorophenyl)-3-methyl-3-(pyrimidin-5-ylmethyl)-1-(1H-1,2,4-triazol-3-yl)-1,3-dihydro-2H-indol-2-one (TROX-1) has an improved therapeutic window compared with ziconotide. Here we characterize TROX-1 inhibition of Cav2.2 channels in more detail. When channels are biased toward open/inactivated states by depolarizing the membrane potential under voltage-clamp electrophysiology, TROX-1 inhibits Ca(V)2.2 channels with an IC(50) of 0.11 μM. The voltage dependence of Ca(V)2.2 inhibition was examined using automated electrophysiology. TROX-1 IC(50) values were 4.2, 0.90, and 0.36 μM at -110, -90, and -70 mV, respectively. TROX-1 displayed use-dependent inhibition of Ca(V)2.2 with a 10-fold IC(50) separation between first (27 μM) and last (2.7 μM) pulses in a train. In a fluorescence-based calcium influx assay, TROX-1 inhibited Ca(V)2.2 channels with an IC(50) of 9.5 μM under hyperpolarized conditions and 0.69 μM under depolarized conditions. Finally, TROX-1 potency was examined across the Ca(V)2 subfamily. Depolarized IC(50) values were 0.29, 0.19, and 0.28 μM by manual electrophysiology using matched conditions and 1.8, 0.69, and 1.1 μM by calcium influx for Ca(V)2.1, Ca(V)2.2, and Ca(V)2.3, respectively. Together, these in vitro data support the idea that a state-dependent, non-subtype-selective Ca(V)2 channel inhibitor can achieve an improved therapeutic window over the relatively state-independent Ca(V)2.2-selective inhibitor ziconotide in preclinical models of chronic pain.

Axonal hyperpolarization in inclusion‐body myopathy, paget disease of the bone, and frontotemporal dementia (IBMPFD)

Inclusion-body myopathy, Paget disease of the bone, and frontotemporal dementia (IBMPFD) is an autosomal dominant disorder due to mutations in the valosin-containing protein (VCP) gene. Patients with this disorder may have neuropathic or myopathic features. Peripheral nerve function and axonal excitability were studied in three members from two families with VCP mutations (p.Arg155Leu and p.Leu198Trp). Patients from the first family had neurogenic patterns on needle electromyography (EMG), whereas those in the second family had myopathic EMG changes. In threshold electrotonus for motor axons, the changes to depolarizing and hyperpolarizing conditioning currents were at or outside control limits in all three patients. Superexcitability was increased, and the relative refractory period was reduced. The strength-duration time constant was normal. In sensory axons of all three patients, there were similar changes in threshold electrotonus, but not in superexcitability. These features are best explained by axonal hyperpolarization. The findings provide insight into the pathophysiological mechanisms in these genotypes and, possibly, into all patients with IBMPFD.

Analgesic Effects of a Substituted N-Triazole Oxindole (TROX-1), a State-Dependent, Voltage-Gated Calcium Channel 2 Blocker

Voltage-gated calcium channel (Ca(v))2.2 (N-type calcium channels) are key components in nociceptive transmission pathways. Ziconotide, a state-independent peptide inhibitor of Ca(v)2.2 channels, is efficacious in treating refractory pain but exhibits a narrow therapeutic window and must be administered intrathecally. We have discovered an N-triazole oxindole, (3R)-5-(3-chloro-4-fluorophenyl)-3-methyl-3-(pyrimidin-5-ylmethyl)-1-(1H-1,2,4-triazol-3-yl)-1,3-dihydro-2H-indol-2-one (TROX-1), as a small-molecule, state-dependent blocker of Ca(v)2 channels, and we investigated the therapeutic advantages of this compound for analgesia. TROX-1 preferentially inhibited potassium-triggered calcium influx through recombinant Ca(v)2.2 channels under depolarized conditions (IC(50) = 0.27 microM) compared with hyperpolarized conditions (IC(50) > 20 microM). In rat dorsal root ganglion (DRG) neurons, TROX-1 inhibited omega-conotoxin GVIA-sensitive calcium currents (Ca(v)2.2 channel currents), with greater potency under depolarized conditions (IC(50) = 0.4 microM) than under hyperpolarized conditions (IC(50) = 2.6 microM), indicating state-dependent Ca(v)2.2 channel block of native as well as recombinant channels. TROX-1 fully blocked calcium influx mediated by a mixture of Ca(v)2 channels in calcium imaging experiments in rat DRG neurons, indicating additional block of all Ca(v)2 family channels. TROX-1 reversed inflammatory-induced hyperalgesia with maximal effects equivalent to nonsteroidal anti-inflammatory drugs, and it reversed nerve injury-induced allodynia to the same extent as pregabalin and duloxetine. In contrast, no significant reversal of hyperalgesia was observed in Ca(v)2.2 gene-deleted mice. Mild impairment of motor function in the Rotarod test and cardiovascular functions were observed at 20- to 40-fold higher plasma concentrations than required for analgesic activities. TROX-1 demonstrates that an orally available state-dependent Ca(v)2 channel blocker may achieve a therapeutic window suitable for the treatment of chronic pain.

Roles of hyperpolarization-activated current If in sinoatrial node pacemaking: insights from bifurcation analysis of mathematical models

To elucidate the roles of hyperpolarization-activated current (I(f)) in sinoatrial node (SAN) pacemaking, we theoretically investigated 1) the effects of I(f) on stability and bifurcation during hyperpolarization of SAN cells; 2) combined effects of I(f) and the sustained inward current (I(st)) or Na(+) channel current (I(Na)) on robustness of pacemaking against hyperpolarization; and 3) whether blocking I(f) abolishes pacemaker activity under certain conditions. Bifurcation analyses were performed for mathematical models of rabbit SAN cells; equilibrium points (EPs), periodic orbits, and their stability were determined as functions of parameters. Unstable steady-state potential region determined with applications of constant bias currents shrunk as I(f) density increased. In the central SAN cell, the critical acetylcholine concentration at which bifurcations, to yield a stable EP and quiescence, occur was increased by smaller I(f), but decreased by larger I(f). In contrast, the critical acetylcholine concentration and conductance of gap junctions between SAN and atrial cells at bifurcations progressively increased with enhancing I(f) in the peripheral SAN cell. These effects of I(f) were significantly attenuated by eliminating I(st) or I(Na), or by accelerating their inactivation. Under hyperpolarized conditions, blocking I(f) abolished SAN pacemaking via bifurcations. These results suggest that 1) I(f) itself cannot destabilize EPs; 2) I(f) improves SAN cell robustness against parasympathetic stimulation via preventing bifurcations in the presence of I(st) or I(Na); 3) I(f) dramatically enhances peripheral cell robustness against electrotonic loads of the atrium in combination with I(Na); and 4) pacemaker activity of hyperpolarized SAN cells could be abolished by blocking I(f).

Mexiletine suppresses nodal persistent sodium currents in sensory axons of patients with neuropathic pain

To investigate changes in axonal persistent Na(+) currents in patients with neuropathic pain and the effects of mexiletine, an analogue of lidocaine, on axonal excitability properties. The technique of latent addition was used to estimate nodal persistent Na(+) currents in superficial radial sensory axons of 17 patients with neuropathic pain/paresthesias before and after mexiletine treatment. Brief hyperpolarizing conditioning currents were delivered, and threshold change at the conditioning-test interval of 0.2 ms was measured as an indicator of the magnitude of persistent Na(+) currents. Threshold changes at 0.2 ms in latent addition were greater in the neuropathic patients than in the normal controls (p<0.001). After mexiletine treatment, there was a reduction in clinical pain scores (p<0.001), associated with decreased threshold changes at 0.2 ms (p<0.001). In patients with neuropathy, nodal persistent Na(+) currents in large sensory fibers increase, and the abnormal currents can be suppressed by mexiletine. Pain reduction after mexiletine treatment raises the possibility that excessive Na(+) currents are also suppressed in small fibers mediating neuropathic pain. Latent addition can be used for indirect in vivo monitoring of nodal Na(+) currents in large sensory fibers, and future studies using this approach in small fibers would provide new insights into the peripheral mechanism of neuropathic pain.

In vivo assessment of HCN channel current (Ih) in human motor axons

The "Trond" protocol of nerve excitability tests has been used widely to assess axonal function in peripheral nerve. In this study, the routine Trond protocol was expanded to refine assessment of cAMP-dependent, hyperpolarization-activated current (I(h)) activity. I(h) activity is generated by hyperpolarization-activated, cyclic nucleotide-modulated (HCN) channels in response to hyperpolarization. It limits activity-dependent hyperpolarization, contributes to neuronal automaticity, and is implicated in chronic pain states. Published data regarding I(h) activity in motor nerve are scant. We used additional strong, prolonged hyperpolarizing conditioning stimuli in the threshold electrotonus component of the Trond protocol to demonstrate the time-course of activation of I(h) in motor axons. Fifteen healthy volunteers were tested on four occasions during 1 week. I(h) action was revealed in the threshold electrotonus by the limiting and often reversal, after about 100 ms, of the threshold increase caused by strong hyperpolarizing currents. Statistical analysis by repeated-measures analysis of variance enabled confidence limits to be established for variation between subjects and within subjects. The results demonstrate that, of all the excitability parameters, those dependent on I(h) were the most characteristic of an individual, because variance between subjects was more than four times the variance within subjects. This study demonstrates a reliable method for in vivo assessment of I(h,) and also serves to document the normal variability in nerve excitability properties within subjects.

A new culturing strategy improves functional neuronal development of human neural progenitor cells

Cell replacement therapies that rely on in vitro differentiation of human neural progenitor cells are a promising strategy to compensate the progressive cell loss in neurodegenerative disorders like Parkinson's disease. We and others observed, that the functional differentiation of progenitors in standard differentiation medium remains limited. The aim of the present study was to optimize neuronal in vitro differentiation by mimicking the physiological shift from depolarizing to hyperpolarizing conditions that occurs during early brain development. Differentiation was initiated using a depolarizing high potassium- and low sodium-containing medium. Subsequently, the high potassium-containing medium was replaced by a hyperpolarizing medium containing low potassium and high sodium concentrations. This two-phase strategy significantly promoted the expression of neuronal markers, enhanced neurite growth, enlarged sodium inward currents, and increased action potential firing. Thus, depolarizing followed by hyperpolarizing culture conditions enable developing human neural progenitor cells to adopt more mature functional qualities.

Ion selectivity of the Kat1 K+channel pore

Kat1 is a highly selective inward-rectifying K(+) channel that opens for extended periods under conditions of extreme hyperpolarization. Over 200 point mutants in the pore region of the Kat1 K(+) channel were generated and examined in the yeast Saccharomyces cerevisiae and Xenopus oocytes to assess the effect of the mutations on ion selectivity. Substitutions at the tyrosine of the signature sequence G-Y-G resulted in the most significant alterations in ion selectivity, consistent with its role in the selectivity filter. However, greater than 80% of the mutations throughout the greater pore region also conferred a defect in selectivity demonstrating that the entire pore of Kat1 contributes to the ion selectivity of this channel. Surprisingly, we identified a novel class of mutant channel that conferred enhanced selectivity of K(+) over Na(+). Mutants of this class frequently displayed sensitivity to the competing ion Cs(+). This finding has led us to speculate that the Kat1 channel pore has evolved to balance not only K(+)/Na(+) selectivity, but selectivity over Cs(+), and possibly a wide spectrum of potential competing ions.

Regional Difference in Dynamical Property of Sinoatrial Node Pacemaking: Role of Na + Channel Current

To elucidate the regional differences in sinoatrial node pacemaking mechanisms, we investigated 1), bifurcation structures during current blocks or hyperpolarization of the central and peripheral cells, 2), ionic bases of regional differences in bifurcation structures, and 3), the role of Na + channel current ( I Na) in peripheral cell pacemaking. Bifurcation analyses were performed for mathematical models of the rabbit sinoatrial node central and peripheral cells; equilibrium points, periodic orbits, and their stability were determined as functions of parameters. Structural stability against applications of acetylcholine or electrotonic modulations of the atrium was also evaluated. Blocking L-type Ca 2+ channel current ( I Ca,L) stabilized equilibrium points and abolished pacemaking in both the center and periphery. Critical acetylcholine concentration and gap junction conductance for pacemaker cessation were higher in the periphery than in the center, being dramatically reduced by blocking I Na. Under hyperpolarized conditions, blocking I Na, but not eliminating I Ca,L, abolished peripheral cell pacemaking. These results suggest that 1), I Ca,L is responsible for basal pacemaking in both the central and peripheral cells, 2), the peripheral cell is more robust in withstanding hyperpolarizing loads than the central cell, 3), I Na improves the structural stability to hyperpolarizing loads, and 4), I Na-dependent pacemaking is possible in hyperpolarized peripheral cells.

Pathophysiologic insights into motor axonal function in Kennedy disease

Kennedy disease (KD), or spinobulbomuscular atrophy, is a slowly progressive inherited neurodegenerative disorder, marked by prominent fasciculations that typically precede the development of other symptoms. Although the genetic basis of KD relates to triplet (CAG) repeat expansion in the androgen receptor (AR) gene on the X chromosome, the mechanisms underlying the clinical presentation in KD have yet to be established. Consequently, the present study applied axonal excitability techniques to investigate the pathophysiologic mechanisms associated with KD. Peripheral nerve excitability studies were undertaken in 7 patients with KD with compound muscle action potentials (CMAP) recorded from the right abductor pollicis brevis. Strength-duration time constant (KD 0.54 +/- 0.03 msec; controls, 0.41 +/- 0.02 msec, p < 0.01) and the hyperpolarizing current/threshold gradient (KD 0.42 +/- 0.01; controls, 0.37 +/- 0.01, p < 0.05) were significantly increased in KD. Strength-duration time constant correlated with the CMAP amplitude (R = 0.68) and the fasciculation frequency (R = 0.62). Threshold electrotonus revealed greater changes in response to subthreshold depolarizing (KD TEd [90 to 100 msec], 50.75 +/- 1.98%; controls TEd [90 to 100 msec], 45.67 +/- 0.67%, p < 0.01) and hyperpolarizing (KD TEh [90 to 100 msec], 128.5 +/- 6.9%; controls TEh [90 to 100 msec], 120.5 +/- 2.4%) conditioning pulses. Measurements of refractoriness, superexcitability, and late subexcitability changed appropriately for axonal hyperpolarization, perhaps reflecting the effects of increased ectopic activity. In total, the increase in the strength-duration time constant may be the primary event, occurring early in course of the disease, contributing to the development of axonal hyperexcitability in Kennedy disease, and thereby to the generation of fasciculations, a characteristic hallmark of the disease.

Increased nodal persistent Na + currents in human neuropathy and motor neuron disease estimated by latent addition

Objective To investigate the changes in nodal persistent Na + currents in human neuropathy and motor neuron disease. In human motor axons, approximately 1.0% of total Na + channels are active at rest, termed “persistent” Na + channels, and the conductance can be non-invasively estimated by the technique of latent addition in vivo. Methods Latent addition was performed in median motor axons of 93 patients with axonal neuropathy ( n = 38), lower motor neuron disorder (LMND; n = 19) or amyotrophic lateral sclerosis (ALS; n = 36) and in 27 age-matched normal subjects. Brief hyperpolarizing conditioning current pulses were delivered, and threshold change at the conditioning-test interval of 0.2 ms was measured as an estimator of the magnitude of persistent Na + currents. Threshold electrotonus and supernormality were also measured as indicators of resting membrane potential. Results Threshold changes at 0.2 ms were significantly greater in patients with neuropathy or LMND ( p < 0.05), and tended to be greater in ALS patients ( p = 0.075) than in normal controls. Threshold electrotonus and supernormality did not differ in each patient group and normal controls, suggesting that membrane potential is not altered in patients. In the recovery phase of axonal neuropathy, the threshold changes increased in parallel with an increase in amplitudes of compound muscle action potential. Conclusions Persistent Na + currents appear to increase commonly in disorders involving lower motor neurons, possibly associated with axonal regeneration or collateral sprouting or changes in Na + channel gating. Significance The increased axonal excitability could partly be responsible for positive motor symptoms such as muscle cramping frequently seen in lower motor neuron disorders.