Overexpressed NaV1.7 Channels Confer Hyperexcitability to in vitro Trigeminal Sensory Neurons of CaV2.1 Mutant Hemiplegic Migraine Mice

Trigeminal sensory neurons of transgenic knock-in (KI) mice expressing the R192Q missense mutation in the α1A subunit of neuronal voltage-gated CaV2.1 Ca2+ channels, which leads to familial hemiplegic migraine type 1 (FHM1) in patients, exhibit a hyperexcitability phenotype. Here, we show that the expression of NaV1.7 channels, linked to pain states, is upregulated in KI primary cultures of trigeminal ganglia (TG), as shown by increased expression of its α1 subunit. In the majority of TG neurons, NaV1.7 channels are co-expressed with ATP-gated P2X3 receptors (P2X3R), which are important nociceptive sensors. Reversing the trigeminal phenotype with selective CaV2.1 channel inhibitor ω-agatoxin IVA inhibited NaV1.7 overexpression. Functionally, KI neurons revealed a TTX-sensitive inward current of larger amplitude that was partially inhibited by selective NaV1.7 blocker Tp1a. Under current-clamp condition, Tp1a raised the spike threshold of both wild-type (WT) and KI neurons with decreased firing rate in KI cells. NaV1.7 activator OD1 accelerated firing in WT and KI neurons, a phenomenon blocked by Tp1a. Enhanced expression and function of NaV1.7 channels in KI TG neurons resulted in higher excitability and facilitated nociceptive signaling. Co-expression of NaV1.7 channels and P2X3Rs in TGs may explain how hypersensitivity to local stimuli can be relevant to migraine.

Transcriptional regulation of intermolecular Ca2+ signaling in hibernating ground squirrel cardiomyocytes: The myocardin–junctophilin axis

The contraction of heart cells is controlled by the intermolecular signaling between L-type Ca2+ channels (LCCs) and ryanodine receptors (RyRs), and the nanodistance between them depends on the interaction between junctophilin-2 (JPH2) in the sarcoplasmic reticulum (SR) and caveolin-3 (CAV3) in the transversal tubule (TT). In heart failure, decreased expression of JPH2 compromises LCC–RyR communication leading to deficient blood-pumping power. In the present study, we found that JPH2 and CAV3 transcription was concurrently regulated by serum response factor (SRF) and myocardin. In cardiomyocytes from torpid ground squirrels, compared with those from euthermic counterparts, myocardin expression was up-regulated, which boosted both JPH2 and CAV3 expression. Transmission electron microscopic imaging showed that the physical coupling between TTs and SRs was tightened during hibernation and after myocardin overexpression. Confocal Ca2+ imaging under the whole-cell patch clamp condition revealed that these changes enhanced the efficiency of LCC–RyR intermolecular signaling and fully compensated the adaptive down-regulation of LCCs, maintaining the power of heart contraction while avoiding the risk of calcium overload during hibernation. Our finding not only revealed an essential molecular mechanism underlying the survival of hibernating mammals, but also demonstrated a “reverse model of heart failure” at the molecular level, suggesting a strategy for treating heart diseases.

Best pattern for locating piezoelectric patches on a plate for maximum critical buckling loads, using particle swarm optimization algorithm

Locating piezoelectric patches on structures under compressive forces as a sensor or actuator can predict or increase the buckling load of the structure. In this article, using particle swarm optimization algorithm, the location and pattern of piezoelectric patches on a plate to achieve maximum buckling load are investigated. For boundary condition of the plate, four different conditions are considered. The piezoelectric patches have more effect on critical buckling load for Clamp-Free condition and less effect for Clamp-Clamp condition. The voltage of patches has a linear effect on increasing the maximum critical buckling load. The results show that locating piezoelectric patches near the clamp edge has more influence on critical load than locating the patches near the simply support or free edge. The results in this article are validated by comparing to the results reported in the previous publication.

Transpulmonary lactate shuttle

The shuttling of intermediary metabolites such as lactate through the vasculature contributes to the dynamic energy and biosynthetic needs of tissues. Tracer kinetic studies offer a powerful tool to measure the metabolism of substrates like lactate that are simultaneously taken up from and released into the circulation by organs, but in each circulatory passage, the entire cardiac output traverses the pulmonary parenchyma. To determine whether transpulmonary lactate shuttling affects whole-body lactate kinetics in vivo, we examined the effects of a lactate load (via lactate clamp, LC) and epinephrine (Epi) stimulation on transpulmonary lactate kinetics in an anesthetized rat model using a primed-continuous infusion of [U-13C]lactate. Under all conditions studied, control 1.2 (SD 0.7) (Con), LC 1.9 (SD 2.5), and Epi 1.9 (SD 3.5) mg/min net transpulmonary lactate uptake occurred. Compared with Con, a lactate load via LC significantly increased mixed central venous ([v̄]) [1.9 mM (SD 0.5) vs. 4.7 (SD 0.4)] and arterial ([a]) [1.6 mM (SD 0.4) vs. 4.1 (SD 0.6)] lactate concentrations ( P < 0.05). Transpulmonary lactate gradient ([v̄] − [a]) was highest during the lactate clamp condition [0.6 mM (SD 0.7)] and lowest during Epi [0.2 mM (SD 0.5)] stimulation ( P < 0.05). Tracer measured lactate fractional extractions were similar for control, 16.6% (SD 15.3), and lactate clamp, 8.2% (SD 15.3) conditions, but negative during Epi stimulation, −25.3% (SD 45.5) when there occurred a transpulmonary production, the conversion of mixed central venous pyruvate to arterial lactate. Further, isotopic equilibration between L and P occurred following tracer lactate infusion, but depending on compartment (v̄ or a) and physiological stimulus, [L]/[P] concentration and isotopic enrichment ratios ranged widely. We conclude that pulmonary arterial-vein concentration difference measurements across the lungs provide an incomplete, and perhaps misleading picture of parenchymal lactate metabolism, especially during epinephrine stimulation.

Systemic administration of anti-NGF increases A-type potassium currents and decreases pancreatic nociceptor excitability in a rat model of chronic pancreatitis

We have previously shown that pancreatic sensory neurons in rats with chronic pancreatitis (CP) display increased excitability associated with a decrease in transient inactivating potassium currents ( IA), thus accounting in part for the hyperalgesia associated with this condition. Because of its well known role in somatic hyperalgesia, we hypothesized a role for the nerve growth factor (NGF) in driving these changes. CP was induced by intraductal injection of trinitrobenzene sulfonic acid (TNBS) in rats. After 3 wk, anti-NGF antibody or control serum was injected intra-peritoneally daily for 1 wk. This protocol was repeated in another set of experiments in control rats (receiving intraductal PBS instead of TNBS). Pancreatic nociceptors labeled with the dye Dil were identified, and patch-clamp recordings were made from acutely dissociated DRG neurons. Sensory neurons from anti-NGF-treated rats displayed a lower resting membrane potential, increased rheobase, decreased burst discharges in response to stimulatory current, and decreased input resistance compared with those treated with control serum. Under voltage-clamp condition, neuronal IA density was increased in anti-NGF-treated rats compared with rats treated with control serum. However, anti-NGF treatment had no effect on electrophysiological parameters in neurons from control rats. The expression of Kv-associated channel or ancillary genes Kv1.4, 4.1, 4.2, 4.3, and DPP6, DPP10, and KCHIPs 1–4 in pancreas-specific nociceptors was examined by laser-capture microdissection and real-time PCR quantification of mRNA levels. No significant differences were seen among those. These findings emphasize a key role for NGF in maintaining neuronal excitability in CP specifically via downregulation of IA by as yet unknown mechanisms.

SPECTRAL ANALYSIS OF CURRENT-NOISE DATA FOR BILAYER LIPID MEMBRANE

We report the spectral analysis of current-noise data obtained under voltage clamp condition in bilayer lipid membrane, using Lomb Scargle Periodogram. Such spectral analysis shows evidence of crossover from an uncorrelated phenomenon towards a state of long range correlation in time and space with the increase of applied bias. Based on these observations a probable mechanism of transmembrane charge conduction has been outlined in the light of self-organized criticality.

Mechanisms Determining the Dynamic Range of the Bullfrog Olfactory Receptor Cell

Spike discharges of single olfactory receptor cells (ORCs) were recorded with the whole cell patch-clamp method applied to slice preparation. In parallel, activities of transduction channels were measured under the voltage-clamp condition. When cells were stimulated by odorants, 54 out of 306 cells exhibited inward current responses (10 mM cineole in the puffer pipette). The amplitude of the inward current was dependent on the stimulus period, reflecting the time integration for the stimulus dose, and the relation could be fitted by the Hill equation. Under the current-clamp condition, current injection induced spike discharges. In cells showing repetitive firings, the firing frequency was dependent on the amount of injected current. The relation was fitted by the Michaelis-Menten equation showing saturation. When cells were responsive to the odorant and had abilities to discharge repetitive spikes, the depolarizing responses were accompanied by repetitive spikes. In those cells, the spike frequency was dose-dependent, expressing saturation similar to the result obtained by current injection. Since both transduction channel and spike generative steps expressed saturation in their dose dependences, we explored what step(s) actually determines saturation in ORC signaling processes. By examining dose-response relations of both the current and spikes in the same cells, saturating dose was found to be dependent largely on that of the transduction step. This suggests that the dynamic range is fundamentally determined by the transduction system. In addition, a simple model derived from the nonlinearity of the plasma membrane could explain that a critical level of dynamic range was, at least in part, modified by the membrane nonlinearity.

Modulation of High-Voltage Activated Ca2+ Channels in the Rat Periaqueductal Gray Neurons by μ-Type Opioid Agonist

Kim, Chang-Ju, Jeong-Seop Rhee, and Norio Akaike. Modulation of high-voltage activated Ca2+ channels in the rat periaqueductal gray neurons by μ-type opioid agonist. J. Neurophysiol. 77: 1418–1424, 1997. The effect of μ-type opioid receptor agonist, D-Ala2,N-MePhe4,Gly5-ol-enkephalin (DAMGO), on high-voltage-activated (HVA) Ca2+ channels in the dissociated rat periaqueductal gray (PAG) neurons was investigated by the use of nystatin-perforated patch recording mode under voltage-clamp condition. Among 118 PAG neurons tested, the HVA Ca2+ channels of 38 neurons (32%) were inhibited by DAMGO (DAMGO-sensitive cells), and the other 80 neurons (68%) were not affected by DAMGO (DAMGO-insensitive cells). The N-, P-, L-, Q-, and R-type Ca2+ channel components in DAMGO-insensitive cells shared 26.9, 37.1, 22.3, 7.9, and 5.8%, respectively, of the total Ca2+ channel current. The channel components of DAMGO-sensitive cells were 45.6, 25.7, 21.7, 4.6, and 2.4%, respectively. The HVA Ca2+ current of DAMGO-sensitive neurons was inhibited by DAMGO in a concentration-, time-, and voltage-dependent manner. Application of ω-conotoxin-GVIA occluded the inhibitory effect of DAMGO ∼70%. So, HVA Ca2+ channels inhibited by DAMGO were mainly the N-type Ca2+ channels. The inhibitory effect of DAMGO on HVA Ca2+ channels was prevented almost completely by the pretreatment of pertussis toxin (PTX) for 8–10 h, suggesting that DAMGO modulation on N-type Ca2+ channels in rat PAG neurons is mediated by PTX-sensitive G proteins. These results indicate that μ-type opioid receptor modulates N-type HVA Ca2+ channels via PTX-sensitive G proteins in PAG neurons of rats.

T-type Ca2+ channel lowers the threshold of spike generation in the newt olfactory receptor cell.

Mechanisms underlying action potential generation in the newt olfactory receptor cell were investigated by using the whole-cell version of the patch-clamp technique. Isolated olfactory cells had a resting membrane potential of -70 +/- 9 mV. Injection of a depolarizing current step triggered action potentials under current clamp condition. The amplitude of the action potential was reduced by lowering external Na+ concentration. After a complete removal of Na+, however, cells still showed action potentials which was abolished either by Ca2+ removal or by an application of Ca2+ channel blocker (Co2+ or Ni2+), indicating an involvement of Ca2+ current in spike generation of newt olfactory receptor cells. Under the voltage clamp condition, depolarization of the cell to -40 mV from the holding voltage of -100 mV induced a fast transient inward current, which consisted of Na+ (INa) and T-type Ca2+ (ICa.T) currents. The amplitude of ICa,T was about one fourth of that of INa. Depolarization to more positive voltages also induced L-type Ca2+ current (ICa,L). ICa,L was as small as a few pA in normal Ringer solution. The activating voltage of ICa,T was approximately 10 mV more negative than that of INa. Under current clamp, action potentials generated by a least effective depolarization was almost completely blocked by 0.1 mM Ni2+ (a specific T-type Ca2+ channel blocker) even in the presence of Na+. These results suggest that ICa,T contributes to action potential in the newt olfactory receptor cell and lowers the threshold of spike generation.