The effects of membrane potential oscillations on the excitability of rat hypoglossal motoneurons.

Oscillations in membrane potential induced by synaptic inputs and intrinsic ion channel activity play a role in regulating neuronal excitability, but the precise mechanisms underlying their contributions remain largely unknown. Here we used electrophysiological and modeling approaches to investigate the effects of Gaussian white noise injected currents on the membrane properties and discharge characteristics of hypoglossal (HG) motoneurons in P16-21 day old rats. We found that the noise-induced membrane potential oscillations facilitated spike initiation by hyperpolarizing the cells' voltage threshold by 3.1 ± 1.0 mV and reducing the recruitment current for the tonic discharges by 0.26 ± 0.1 nA, on average (n = 59). Further analysis revealed that the noise reduced both recruitment and decruitment currents by 0.26 ± 0.13 and 0.33 ± 0.1 nA, respectively, and prolonged the repetitive firing. The noise also increased the slopes of frequency-current (F-I) relationships by 1.1 ± 0.2 Hz/nA. To investigate the potential mechanisms underlying these findings, we constructed a series of HG motoneuron models based on their electrophysiological properties. The models consisted of five compartments endowed with transient sodium (NaT), delayed-rectify potassium [K(DR)], persistent sodium (NaP), calcium-activated potassium [K(AHP)], L-type calcium (CaL) and H-current channels. In general, all our experimental results could be well fitted by the models, however, a modification of standard Hodgkin-Huxley kinetics was required to reproduce the changes in the F-I relationships and the prolonged discharge firing. This modification, corresponding to the noise generated by the stochastic flicker of voltage-gated ion channels (channel flicker, CF), was an adjustable sinusoidal function added to kinetics of the channels that increased their sensitivity to subthreshold membrane potential oscillations. Models with CF added to NaP and CaL channels mimicked the noise-induced alterations of membrane properties, whereas models with CF added to NaT and K(DR) were particularly effective in reproducing the noise-induced changes for repetitive firing observed in the real motoneurons. Further analysis indicated that the modified channel kinetics enhanced NaP- and CaL-mediated inward currents thus increasing the excitability and output of HG motoneurons, whereas they produced relatively small changes in NaT and K(DR), thus balancing these two currents and triggering variability of repetitive firing. This study provided insight into the types of membrane channel mechanisms that might underlie oscillation-induced alterations of neuronal excitability and motor output in rat HG motoneurons.

Mechanisms and physiological implications of cooperative gating of clustered ion channels.

Ion channels play a central role in the regulation of nearly every cellular process. Dating back to the classic 1952 Hodgkin-Huxley model of the generation of the action potential, ion channels have always been thought of as independent agents. A myriad of recent experimental findings exploiting advances in electrophysiology, structural biology, and imaging techniques, however, have posed a serious challenge to this long-held axiom, as several classes of ion channels appear to open and close in a coordinated, cooperative manner. Ion channel cooperativity ranges from variable-sized oligomeric cooperative gating in voltage-gated, dihydropyridine-sensitive CaV1.2 and CaV1.3 channels to obligatory dimeric assembly and gating of voltage-gated NaV1.5 channels. Potassium channels, transient receptor potential channels, hyperpolarization cyclic nucleotide-activated channels, ryanodine receptors (RyRs), and inositol trisphosphate receptors (IP3Rs) have also been shown to gate cooperatively. The implications of cooperative gating of these ion channels range from fine-tuning excitation-contraction coupling in muscle cells to regulating cardiac function and vascular tone, to modulation of action potential and conduction velocity in neurons and cardiac cells, and to control of pacemaking activity in the heart. In this review, we discuss the mechanisms leading to cooperative gating of ion channels, their physiological consequences, and how alterations in cooperative gating of ion channels may induce a range of clinically significant pathologies.

Excessive Homeostatic Gain in Spinal Motoneurons in a Mouse Model of Amyotrophic Lateral Sclerosis.

In the mSOD1 model of ALS, the excitability of motoneurons is poorly controlled, oscillating between hyperexcitable and hypoexcitable states during disease progression. The hyperexcitability is mediated by excessive activity of voltage-gated Na+ and Ca2+ channels that is initially counteracted by aberrant increases in cell size and conductance. The balance between these opposing actions collapses, however, at the time that the denervation of muscle fibers begins at about P50, resulting in a state of hypo-excitability and cell death. We propose that this process of neurodegeneration ensues from homeostatic dysregulation of excitability and have tested this hypothesis by perturbing a signal transduction pathway that plays a major role in controlling biogenesis and cell size. Our 『homeostatic dysregulation hypothesis' predicted that neonatal mSOD1 motoneurons would be much more sensitive to such perturbations than wild type controls and our results strongly support this hypothesis. Our results have important implications for therapeutic approaches to ALS.

Nonlinear Input-Output Functions of Motoneurons.

All movements are generated by the activation of motoneurons, and hence their input-output properties define the final step in processing of all motor commands. A major challenge to understanding this transformation has been the striking nonlinear behavior of motoneurons conferred by the activation of persistent inward currents (PICs) mediated by their voltage-gated Na+ and Ca2+ channels. In this review, we focus on the contribution that these PICs make to motoneuronal discharge and how the nonlinearities they engender impede the construction of a comprehensive model of motor control.

A stochastic model of ion channel cluster formation in the plasma membrane.

Ion channels are often found arranged into dense clusters in the plasma membranes of excitable cells, but the mechanisms underlying the formation and maintenance of these functional aggregates are unknown. Here, we tested the hypothesis that channel clustering is the consequence of a stochastic self-assembly process and propose a model by which channel clusters are formed and regulated in size. Our hypothesis is based on statistical analyses of the size distributions of the channel clusters we measured in neurons, ventricular myocytes, arterial smooth muscle, and heterologous cells, which in all cases were described by exponential functions, indicative of a Poisson process (i.e., clusters form in a continuous, independent, and memory-less fashion). We were able to reproduce the observed cluster distributions of five different types of channels in the membrane of excitable and tsA-201 cells in simulations using a computer model in which channels are "delivered" to the membrane at randomly assigned locations. The model's three parameters represent channel cluster nucleation, growth, and removal probabilities, the values of which were estimated based on our experimental measurements. We also determined the time course of cluster formation and membrane dwell time for CaV1.2 and TRPV4 channels expressed in tsA-201 cells to constrain our model. In addition, we elaborated a more complex version of our model that incorporated a self-regulating feedback mechanism to shape channel cluster formation. The strong inference we make from our results is that CaV1.2, CaV1.3, BK, and TRPV4 proteins are all randomly inserted into the plasma membranes of excitable cells and that they form homogeneous clusters that increase in size until they reach a steady state. Further, it appears likely that cluster size for a diverse set of membrane-bound proteins and a wide range of cell types is regulated by a common feedback mechanism.

Ca(2+) entry into neurons is facilitated by cooperative gating of clustered CaV1.3 channels.

CaV1.3 channels regulate excitability in many neurons. As is the case for all voltage-gated channels, it is widely assumed that individual CaV1.3 channels behave independently with respect to voltage-activation, open probability, and facilitation. Here, we report the results of super-resolution imaging, optogenetic, and electrophysiological measurements that refute this long-held view. We found that the short channel isoform (CaV1.3S), but not the long (CaV1.3L), associates in functional clusters of two or more channels that open cooperatively, facilitating Ca(2+) influx. CaV1.3S channels are coupled via a C-terminus-to-C-terminus interaction that requires binding of the incoming Ca(2+) to calmodulin (CaM) and subsequent binding of CaM to the pre-IQ domain of the channels. Physically-coupled channels facilitate Ca(2+) currents as a consequence of their higher open probabilities, leading to increased firing rates in rat hippocampal neurons. We propose that cooperative gating of CaV1.3S channels represents a mechanism for the regulation of Ca(2+) signaling and electrical activity.

Graded Ca²âº/calmodulin-dependent coupling of voltage-gated CaV1.2 channels.

In the heart, reliable activation of Ca(2+) release from the sarcoplasmic reticulum during the plateau of the ventricular action potential requires synchronous opening of multiple CaV1.2 channels. Yet the mechanisms that coordinate this simultaneous opening during every heartbeat are unclear. Here, we demonstrate that CaV1.2 channels form clusters that undergo dynamic, reciprocal, allosteric interactions. This 'functional coupling' facilitates Ca(2+) influx by increasing activation of adjoined channels and occurs through C-terminal-to-C-terminal interactions. These interactions are initiated by binding of incoming Ca(2+) to calmodulin (CaM) and proceed through Ca(2+)/CaM binding to the CaV1.2 pre-IQ domain. Coupling fades as [Ca(2+)]i decreases, but persists longer than the current that evoked it, providing evidence for 'molecular memory'. Our findings suggest a model for CaV1.2 channel gating and Ca(2+)-influx amplification that unifies diverse observations about Ca(2+) signaling in the heart, and challenges the long-held view that voltage-gated channels open and close independently.

Soma size and Cav1.3 channel expression in vulnerable and resistant motoneuron populations of the SOD1G93A mouse model of ALS.

Although the loss of motoneurons is an undisputed feature of amyotrophic lateral sclerosis (ALS) in man and in its animal models (SOD1 mutant mice), how the disease affects the size and excitability of motoneurons prior to their degeneration is not well understood. This study was designed to test the hypothesis that motoneurons in mutant SOD1(G93A) mice exhibit an enlargement of soma size (i.e., cross-sectional area) and an increase in Cav1.3 channel expression at postnatal day 30, well before the manifestation of physiological symptoms that typically occur at p90 (Chiu et al. 1995). We made measurements of spinal and hypoglossal motoneurons vulnerable to degeneration, as well as motoneurons in the oculomotor nucleus that are resistant to degeneration. Overall, we found that the somata of motoneurons in male SOD1(G93A) mutants were larger than those in wild-type transgenic males. When females were included in the two groups, significance was lost. Expression levels of the Cav1.3 channels were not differentiated by genotype, sex, or any interaction of the two. These results raise the intriguing possibility of an interaction between male sex steroid hormones and the SOD1 mutation in the etiopathogenesis of ALS.

Context-dependent coding in single neurons.

The linear-nonlinear cascade model (LN model) has proven very useful in representing a neural system's encoding properties, but has proven less successful in reproducing the firing patterns of individual neurons whose behavior is strongly dependent on prior firing history. While the cell's behavior can still usefully be considered as feature detection acting on a fluctuating input, some of the coding capacity of the cell is taken up by the increased firing rate due to a constant "driving" direct current (DC) stimulus. Furthermore, both the DC input and the post-spike refractory period generate regular firing, reducing the spike-timing entropy available for encoding time-varying fluctuations. In this paper, we address these issues, focusing on the example of motoneurons in which an afterhyperpolarization (AHP) current plays a dominant role regularizing firing behavior. We explore the accuracy and generalizability of several alternative models for single neurons under changes in DC and variance of the stimulus input. We use a motoneuron simulation to compare coding models in neurons with and without the AHP current. Finally, we quantify the tradeoff between instantaneously encoding information about fluctuations and about the DC.

Graft-versus-host disease prophylaxis with everolimus and tacrolimus is associated with a high incidence of sinusoidal obstruction syndrome and microangiopathy: results of the EVTAC trial.

A calcineurin inhibitor combined with methotrexate is the standard prophylaxis for graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (HSCT). Everolimus, a derivative of sirolimus, seems to mediate antileukemia effects. We report on a combination of everolimus and tacrolimus in 24 patients (median age, 62 years) with either myelodysplastic syndrome (MDS; n = 17) or acute myeloid leukemia (AML; n = 7) undergoing intensive conditioning followed by HSCT from related (n = 4) or unrelated (n = 20) donors. All patients engrafted, and only 1 patient experienced grade IV mucositis. Nine patients (37%) developed acute grade II-IV GVHD, and 11 of 17 evaluable patients (64%) developed chronic extensive GVHD. Transplantation-associated microangiopathy (TMA) occurred in 7 patients (29%), with 2 cases of acute renal failure. The study was terminated prematurely because an additional 6 patients (25%) developed sinusoidal obstruction syndrome (SOS), which was fatal in 2 cases. With a median follow-up of 26 months, the 2-year overall survival rate was 47%. Although this new combination appears to be effective as a prophylactic regimen for acute GVHD, the incidence of TMA and SOS is considerably higher than seen with other regimens.

Facilitation of somatic calcium channels can evoke prolonged tail currents in rat hypoglossal motoneurons.

Voltage-dependent persistent inward currents (PICs) make an important contribution to the input-output properties of alpha motoneurons. PICs are thought to be mediated by membrane channels located primarily on the dendrites as evidenced by prolonged tail currents following the termination of a voltage step and by a clockwise hysteresis in the whole cell inward currents recorded in response to depolarizing then repolarizing voltage ramp commands. We report here, however, that voltage-clamp currents with these same features can be generated in isolated somatic membrane patches from rat hypoglossal motoneurons. Long-lasting (200-800 ms) tail currents after 1-s voltage-clamp pulses were observed in nucleated patches from 16 of 23 cells. Further, these somatic PICs display "facilitation" in response to conditioning depolarization as previously observed in whole cell recordings from intact neurons. Pharmacological tests suggest that the PICs were primarily mediated by Cav1 channels. Our results show that many of the features of persistent calcium currents recorded from intact motoneurons do not necessarily reflect a remote dendritic origin but can also be ascribed to the intrinsic properties of their Cav1 channels.

Contribution of persistent sodium currents to spike-frequency adaptation in rat hypoglossal motoneurons.

In response to constant current inputs, the firing rates of motoneurons typically show a continuous decline over time. The biophysical mechanisms underlying this process, called spike-frequency adaptation, are not well understood. Spike-frequency adaptation normally exhibits a rapid initial phase, followed by a slow, later phase that continues throughout the duration of firing. One possible mechanism mediating the later phase might be a reduction in the persistent sodium current (I(NaP)) that has been shown to diminish the capacity of cortical pyramidal neurons and spinal motoneurons to sustain repetitive firing. In this study, we used the anticonvulsant phenytoin to reduce the I(NaP) of juvenile rat hypoglossal motoneurons recorded in brain stem slices, and we examined the consequences of a reduction in I(NaP) on the magnitude and time course of spike-frequency adaptation. Adding phenytoin to the bathing solution (> or =50 microM) generally produced a marked reduction in the persistent inward currents (PICs) recorded at the soma in response to slow, voltage-clamp triangular ramp commands (-70 to 0 mV and back). However, the same concentrations of phenytoin appeared to have no significant effect on spike-frequency adaptation even though the phenytoin often augmented the reduction in action potential amplitude that occurs during repetitive firing. The surprising finding that the reduction of a source of sustained inward current had no appreciable effect on the pattern of spike generation suggests that several types of membrane channels must act cooperatively to insure that these motoneurons can generate the sustained repetitive firing required for long-lasting motor behaviors.

Persistent sodium and calcium currents in rat hypoglossal motoneurons.

Voltage-dependent persistent inward currents are thought to make an important contribution to the input-output properties of alpha-motoneurons, influencing both the transfer of synaptic current to the soma and the effects of that current on repetitive discharge. Recent studies have paid particular attention to the contribution of L-type calcium channels, which are thought to be widely distributed on both the somatic and the dendritic membrane. However, the relative contribution of different channel subtypes as well as their somatodendritic distribution may vary among motoneurons of different species, developmental stages, and motoneuron pools. In this study, we have characterized persistent inward currents in juvenile (10- to 24-day-old) rat hypoglossal (HG) motoneurons. Whole-cell, voltage-clamp recordings were made from the somata of visualized rat HG motoneurons in 300-microm brain stem slices. Slow (10 s), triangular voltage-clamp commands from a holding potential of -70 to 0 mV and back elicited whole-cell currents that were dominated by outward, potassium currents, but often showed a region of negative slope resistance on the rising phase of the command. In the presence of potassium channel blockers (internal cesium and external 4-aminopyridine and tetraethylammonium), net inward currents were present on both the rising and falling phases of the voltage-clamp command. A portion of the inward current present on the ascending phase of the command was mediated by TTX-sensitive sodium channels, whereas calcium channels mediated the remainder of the current. We found roughly the same relative contributions of P-, N-, and L-type channels to the calcium currents recorded at the soma that had previously been found in neonatal rat HG motoneurons. In most cells, the somatic voltage thresholds for calcium current onset and offset were similar and the peak current was largest on the ascending phase of the clamp command. However, about one-third of the cells exhibited a substantial clockwise current hysteresis, i.e., inward currents were present at lower voltages on the descending phase of the clamp command. In the same cells, 1-s depolarizing voltage-clamp commands were followed by prolonged tail currents, consistent with a prominent contribution from dendritic channels. In contrast to previous reports on turtle and mouse motoneurons, blocking L-type calcium channels did not eliminate these presumed dendritic currents.

Individual hymenoptera venom compounds induce upregulation of the basophil activation marker ectonucleotide pyrophosphatase/phosphodiesterase 3 (CD203c) in sensitized patients.

BACKGROUND: Bee and wasp venom extracts contain potent allergens capable of inducing severe clinical reactions. To analyze immediate-type hypersensitivity to defined hymenoptera venom components, a recently developed in vitro test was applied that is based on the upregulation of CD203c expression on basophils. METHODS: CD203c expression on blood basophils of 9 healthy donors and 39 patients allergic to bee and/or wasp venom was analyzed by flow cytometry before and after activation with the purified bee venom allergens phospholipase A2 (Api m 1), hyaluronidase (Api m 2) and melittin (Api m 4), or the purified wasp venom allergens phospholipase A1 (Ves v 1), hyaluronidase (Ves v 2) and the recombinant antigen 5 (Ves v 5). Venom-induced CD203c upregulation on basophils was compared with skin tests and assessment of specific IgE. Basophils of nonresponders were preincubated with 10 ng/ml interleukin-3 (IL-3) prior to allergen stimulation. RESULTS: CD203c upregulation on basophils was induced by defined hymenoptera venom components in 35/39 patients with a diagnosed allergy to wasp and/or bee venom. Twenty-seven of the 34 tested patients with wasp allergy showed CD203c upregulation in response to Ves v 5, 26 of these patients also reacted with Ves v 2 and 17 with Ves v 1. Nine of 13 patients with bee allergy reacted with Api m 1, 13 individuals with Api m 2 and none of these patients with the minor allergen Api m 4. A diagnosed wasp allergy could also be confirmed in the prestimulated basophils (IL-3) of 2 nonresponder individuals who failed to upregulate CD203c in response to IgE receptor cross-linking prior to culture with IL-3. CONCLUSIONS: Flow-cytometric determination of CD203c upregulation on basophils activated by molecularly defined allergens is a powerful method to identify the precise allergen reactivity in sensitized individuals.

What can be learned about motoneurone properties from studying firing patterns?

The discharge patterns of tonically-firing neurones are influenced by both the characteristics of their presynaptic input and their intrinsic properties. The regularity of the discharge of motoneurones is thought to reflect their prominent post-spike afterhyperpolarization (AHP). When a motoneurone fires at steady mean rate, the distribution of interspike intervals is determined by the amplitude and frequency content of the synaptic noise together with the decrease in excitability following a spike due to AHP. This paper considers how motoneurone discharge statistics can be used to estimate AHP trajectories as well as a motoneurone's sensitivity to excitatory input.

Relative strengths and distributions of different sources of synaptic input to the motoneurone pool: implications for motor unit recruitment.

Understanding how synaptic inputs from segmental and descending systems shape motor output from the spinal cord requires information on the relative magnitudes of the synaptic currents produced by the different systems and their patterns of distribution within a motoneurone pool. Equally important are quantitative descriptions of how different synaptic inputs are integrated when they are concurrently active and of how voltage- and ligand-gated conductances on the dendrites of motoneurones affect the transfer of synaptic currents to the soma. We have carried out a number of experimental studies of these inter-related problems on motoneurones in the cat spinal cord and have explored the implications of our findings with computer simulations utilizing a synthetic model of the cat medial gastrocnemius motoneurone pool. This paper provides a brief review of the principal results of our studies.

Integration of synaptic and intrinsic dendritic currents in cat spinal motoneurons.

The results of recent studies designed to reveal some of the 'rules' governing the integration of synaptic and intrinsic dendritic currents in spinal motoneurons are reviewed. When two or more sources of synaptic input are activated concurrently, their combined postsynaptic effects on cat spinal motoneurons with 'passive dendrites' are generally equal to or slightly less than those predicted from the linear sum of their individual effects. However, for experimental preparations in which active conductances on motoneuron dendrites are enabled, instances of greater-than- or less-than linear summation can occur. Further, these studies demonstrate that the persistent inward currents that are generated by motoneuron dendrites provide an intrinsic source of excitatory drive that is larger than those associated with any of the individual synaptic input systems studied to date. Since these intrinsic depolarizing currents can be rapidly inactivated by a hyperpolarizing input, they are ideally suited to providing a major source of the alternating 'drive' to motoneurons during locomotion.