Changes In Action Potential Properties Research Articles

GluN2A or GluN2B subunits of the NMDA receptor contribute to changes in neuronal excitability and impairments in LTP in the hippocampus of aging mice but do not mediate detrimental effects of oligomeric Aβ (1–42)

Studies in rodent models have revealed that oligomeric beta-amyloid protein [Aβ (1–42)] plays an important role in the pathogenesis of Alzheimer’s disease. Early elevations in hippocampal neuronal excitability caused by Aβ (1–42) have been proposed to be mediated via enhanced activation of GluN2B-containing N-methyl-D-aspartate receptors (NMDAR). To what extent GluN2A or GluN2B-containing NMDAR contribute to Aβ (1–42)-mediated impairments of hippocampal function in advanced rodent age is unclear. Here, we assessed hippocampal long-term potentiation (LTP) and neuronal responses 4–5 weeks after bilateral intracerebral inoculation of 8–15 month old GluN2A+/− or GluN2B+/− transgenic mice with oligomeric Aβ (1–42), or control peptide. Whole-cell patch-clamp recordings in CA1 pyramidal neurons revealed a more positive resting membrane potential and increased total spike time in GluN2A+/−, but not GluN2B+/−-hippocampi following treatment with Aβ (1–42) compared to controls. Action potential 20%-width was increased, and the descending slope was reduced, in Aβ–treated GluN2A+/−, but not GluN2B+/− hippocampi. Sag ratio was increased in Aβ–treated GluN2B+/−-mice. Firing frequency was unchanged in wt, GluN2A+/−, and GluN2B+/−hippocampi after Aβ–treatment. Effects were not significantly different from responses detected under the same conditions in wt littermates, however. LTP that lasted for over 2 h in wt hippocampal slices was significantly reduced in GluN2A+/− and was impaired for 15 min in GluN2B+/−-hippocampi compared to wt littermates. Furthermore, LTP (>2 h) was significantly impaired in Aβ–treated hippocampi of wt littermates compared to wt treated with control peptide. LTP induced in Aβ–treated GluN2A+/− and GluN2B+/−-hippocampi was equivalent to LTP in control peptide-treated transgenic and Aβ–treated wt animals. Taken together, our data indicate that knockdown of GluN2A subunits subtly alters membrane properties of hippocampal neurons and reduces the magnitude of LTP. GluN2B knockdown reduces the early phase of LTP but leaves later phases intact. Aβ (1–42)-treatment slightly exacerbates changes in action potential properties in GluN2A+/−-mice. However, the vulnerability of the aging hippocampus to Aβ–mediated impairments of LTP is not mediated by GluN2A or GluN2B-containing NMDAR.

Abstract 16939: Metabolic and Electrophysiological Alterations Underlie Proarrhythmic Properties of Highly Reactive Isolevuglandins in Atrial Cardiomyocytes

Background: Hypertension is one of the most common risk factors for atrial fibrillation (AF), although the precise cellular and molecular mechanism(s) by which hypertension leads to AF are not well understood. Isolevuglandins (IsoLGs) are highly reactive dicarbonyl products of lipid peroxidation responsible for a major component of oxidative stress-related injury. In a mouse model of hypertension, we recently demonstrated that IsoLGs are elevated in hypertensive mouse atria and that an IsoLG scavenger reduced both IsoLG burden and AF susceptibility. Hypothesis: In this study, we hypothesized that IsoLGs can promote AF by inducing proarrhythmic metabolic and electrophysiologic (EP) changes in atrial cardiomyocytes. Methods and Results: Using standard patch clamp methods, we found significant changes in action potential properties of isolated mouse atrial cardiomyocytes exposed to IsoLGs (1μM, n=15 cells), including elevation of resting membrane potential, shortening of APD and reduction of V max . Acute IsoLG treatment led to a reduction of intracellular ATP production in atrial HL-1 cardiomyocytes, as measured by using a luminescence assay. Employing TMRM and Mitotracker Green staining for confocal and high-throughput screening (HTS) live-cell imaging assays, we also found that IsoLGs decreased mitochondrial membrane potential (compared to control, TMRM fluorescence decreased by 23%, 28%, 36% and 42%, respectively, when exposed to 0.01, 0.1, 0.5 and 1μM concentrations of IsoLG) accompanied by increased apoptosis (Cell Event Caspase-3/7 Green Detection Reagent) in a concentration-dependent manner, suggesting a prolonged mitochondrial transition pore opening. Moreover, cell metabolism assays performed using Agilent’s Seahorse XF96 extracellular flux analyzer revealed that IsoLGs exert a concentration dependent decrease in basal oxygen consumption rate and ATP production in HL-1 atrial cardiomyocytes. Conclusion: Together, these findings indicate that IsoLGs promote proarrhythmic EP and mitochondrial effects in atrial cells and thus may provide a novel therapeutic target for AF.

Sex-dependent alterations in the physiology of entorhinal cortex neurons in old heterozygous 3xTg-AD mice

While the higher prevalence of Alzheimer’s disease (AD) in women is clear, studies suggest that biological sex may also influence AD pathogenesis. However, mechanisms behind these differences are not clear. To investigate physiological differences between sexes at the cellular level in the brain, we investigated the intrinsic and synaptic properties of entorhinal cortex neurons in heterozygous 3xTg-AD mice of both sexes at the age of 20 months. This brain region was selected because of its early association with AD symptoms. First, we found physiological differences between male and female non-transgenic mice, providing indirect evidence of axonal alterations in old females. Second, we observed a transgene-dependent elevation of the firing activity, post-burst afterhyperpolarization (AHP), and spontaneous excitatory postsynaptic current (EPSC) activity, without any effect of sex. Third, the passive properties and the hyperpolarization-activated current (Ih) were altered by transgene expression only in female mice, whereas the paired-pulse ratio (PPR) of evoked EPSC was changed only in males. Fourth, both sex and transgene expression were associated with changes in action potential properties. Consistent with previous work, higher levels of Aβ neuropathology were detected in 3xTg-AD females, whereas tau deposition was similar. In summary, our results support the idea that aging and AD neuropathology differentially alter the physiology of entorhinal cortex neurons in males and females.

Differences in electrophysiological properties of functionally identified nociceptive sensory neurons in an animal model of cancer-induced bone pain.

BackgroundBone cancer pain is often severe, yet little is known about mechanisms generating this type of chronic pain. While previous studies have identified functional alterations in peripheral sensory neurons that correlate with bone tumours, none has provided direct evidence correlating behavioural nociceptive responses with properties of sensory neurons in an intact bone cancer model.ResultsIn a rat model of prostate cancer-induced bone pain, we confirmed tactile hypersensitivity using the von Frey test. Subsequently, we recorded intracellularly from dorsal root ganglion neurons in vivo in anesthetized animals. Neurons remained connected to their peripheral receptive terminals and were classified on the basis of action potential properties, responses to dorsal root stimulation, and to mechanical stimulation of the respective peripheral receptive fields. Neurons included C-, Aδ-, and Aβ-fibre nociceptors, identified by their expression of substance P. We suggest that bone tumour may induce phenotypic changes in peripheral nociceptors and that these could contribute to bone cancer pain.ConclusionsThis work represents a significant technical and conceptual advance in the study of peripheral nociceptor functions in the development of cancer-induced bone pain. This is the first study to report that changes in sensitivity and excitability of dorsal root ganglion primary afferents directly correspond to mechanical allodynia and hyperalgesia behaviours following prostate cancer cell injection into the femur of rats. Furthermore, our unique combination of techniques has allowed us to follow, in a single neuron, mechanical pain-related behaviours, electrophysiological changes in action potential properties, and dorsal root substance P expression. These data provide a more complete understanding of this unique pain state at the cellular level that may allow for future development of mechanism-based treatments for cancer-induced bone pain.

Abstract 15625: Flecainide Modulates Action Potential Properties of Cardiac Sympathetic Neurons

Introduction: Sympathetic nerve activity is important in the genesis of catecholaminergic polymorphic ventricular tachycardia (CPVT). Flecainide is effective in managing patients with CPVT. Hypothesis: We hypothesize that flecainide in clinically relevant concentrations alters electrical properties of cardiac sympathetic nerves. Methods: We used the perforated-patch technique in current-clamp mode to record action potentials from single sympathetic neurons isolated from stellate and superior cervical ganglia of ∼2-month-old mice. Action potentials in response to current pulses (10 ms, 0.05 to 0.2 nA, 0.2 Hz) were acquired in control, 1, 5, 10 μM flecainide, and wash out. Results: Flecainide dose-dependently decreased action potential amplitude (in mV, medians with 25 and 75% percentiles) from 131.6 [127.0 - 138.0; control] to 131.1 [123.7 - 137.5; 1 μM], 128.9 [120.0 - 134.4; 5 μM] and 126.1 [115.9 - 132.6; 10 μM ; P < 0.05 by Repeated Measures ANOVA], and upstroke dV/dt max (in V/s) from 172 [109 - 216; control] to 158 [110 - 198; 1 μM), 109 [98 - 174; 5 μM) and 92 [84 - 147; 10 μM; P < 0.05]. Action potential duration at 50% repolarization was significantly prolonged at 10 μM flecainide ( P < 0.05). The changes were reversible upon washout. Resting membrane potential was unaltered. The Figure shows representative action potentials for control and 10 μM flecainide. Conclusions: Flecainide-induced changes in action potential properties are compatible with inhibition of neuron-type voltage-gated Na + channels and K + channels. These actions may decrease sympathetic excitability on the heart, thus help control CPVT.

Decreased cardiac excitability secondary to reduction of sodium current may be a significant contributor to reduced contractility in a rat model of sepsis

IntroductionMultisystem organ failure remains a poorly understood complication of sepsis. During sepsis, reduced excitability contributes to organ failure of skeletal muscle, nerves and the spinal cord. The goal of this study was to determine whether reduced excitability might also contribute to cardiac failure during sepsis.MethodsWistar rats were made septic by cecal ligation and puncture. One day later, action potentials were recorded from beating left ventricular papillary muscle ex vivo by impaling myocytes with sharp microelectrodes.ResultsIn cardiac papillary muscle from septic rats, action potential amplitude and rate of rise were reduced, while threshold was elevated. These changes in action potential properties suggest sepsis selectively reduces sodium current. To determine the effects of selective reduction in sodium current, we applied tetrodotoxin to papillary muscle from healthy rats and found reduction in action potential amplitude and rate of rise, as well as elevation of threshold. The changes were similar to those triggered by sepsis. Blocking calcium current using nifedipine did not mimic action potential changes induced by sepsis. Contractility of healthy papillary muscle was reduced to 40% of normal following partial block of sodium current by tetrodotoxin, close to the low contractility of septic papillary muscle, which was 30% of normal.ConclusionsOur data suggest cardiac excitability is reduced during sepsis in rats. The reduction in excitability appears to be primarily due to reduction of sodium current. The reduction in sodium current may be sufficient to explain most of the reduction in cardiac contractility during sepsis.

Pituitary Adenylate Cyclase 1 Receptor Internalization and Endosomal Signaling Mediate the Pituitary Adenylate Cyclase Activating Polypeptide-Induced Increase in Guinea Pig Cardiac Neuron Excitability

After G-protein-coupled receptor activation and signaling at the plasma membrane, the receptor complex is often rapidly internalized via endocytic vesicles for trafficking into various intracellular compartments and pathways. The formation of signaling endosomes is recognized as a mechanism that produces sustained intracellular signals that may be distinct from those generated at the cell surface for cellular responses including growth, differentiation, and survival. Pituitary adenylate cyclase activating polypeptide (PACAP; Adcyap1) is a potent neurotransmitter/neurotrophic peptide and mediates its diverse cellular functions in part through internalization of its cognate G-protein-coupled PAC1 receptor (PAC1R; Adcyap1r1). In the present study, we examined whether PAC1R endocytosis participates in the regulation of neuronal excitability. Although PACAP increased excitability in 90% of guinea pig cardiac neurons, pretreatment with Pitstop 2 or dynasore to inhibit clathrin and dynamin I/II, respectively, suppressed the PACAP effect. Subsequent addition of inhibitor after the PACAP-induced increase in excitability developed gradually attenuated excitability with no changes in action potential properties. Likewise, the PACAP-induced increase in excitability was markedly decreased at ambient temperature. Receptor trafficking studies with GFP-PAC1 cell lines demonstrated the efficacy of Pitstop 2, dynasore, and low temperatures at suppressing PAC1R endocytosis. In contrast, brefeldin A pretreatments to disrupt Golgi vesicle trafficking did not blunt the PACAP effect, and PACAP/PAC1R signaling still increased neuronal cAMP production even with endocytic blockade. Our results demonstrate that PACAP/PAC1R complex endocytosis is a key step for the PACAP modulation of cardiac neuron excitability.

Abstract 474: Impaired Excitability of Renal Afferent Innervation in an in-vitro Model of Inflammation.

INTRODUCTION AND AIMS: Renal denervation may be beneficial in hypertension with inflammatory renal disease: in hypertension renal afferent control of sympathetic nerve activity could be impaired. Recently, we found that afferent renal neurons showed a characteristic excitability, exhibiting predominantly a sustained firing upon current injection. So far, excitability of these neurons under pathological conditions such as renal inflammation in hypertension is unclear. Hence, in an in vitro model we wanted to test the hypothesis that a proinflammatory mediator like CXCL1 could alter the firing pattern of neurons with renal afferents and thus decrease their excitability. METHODS: Dorsal root ganglion (DRG) neurons (Th11-L2) were incubated with the chemokine CXCL1 (1,5nmol/ml) for 12 hours before patch clamp recordings. Labelling (DiI) allowed the identification of renal afferent neurons. Current clamp was used to characterize neurons as “tonic”, i.e. sustained action potential (AP) firing or “phasic”, i.e. <5 APs according to their firing response to current injections. AP properties were determined in renal and non-renal neurons incubated with CXCL1 and compared to controls. Results: Renal afferent DRG neurons exhibited in 57% (23 of 41) a tonic firing pattern vs.11,7% (3 of 27)* in non-renal neurons. However, exposed to the chemokine, renal DRG neurons exhibited significantly to a lesser degree tonic firing (35,6% [35 of 101] vs. 57% [23 of 41], *p<0,05) but instead an increased occurrence of phasic firing. Renal DRG neurons with phasic firing pattern showed a significantly lower threshold for AP-firing (600pA [320-1000] vs.1000pA [400-3200]) and a significantly shorter AP-duration at threshold-level (2,095ms [1,75-4,25] vs. 5,15ms [4,3- 8,7]) after exposure to CXCL1. Conclusion: We could show that after exposure to a proinflammatory mediator like CXCL1, renal afferent DRG neurons exhibited a significantly higher proportion of neurons with a phasic pattern (<5 APs) and decreased excitability as compared to control conditions. Significant changes in action potential properties of phasic neurons point to an altered sodium channel expression likely inducing a faster inactivation of these channels with decreased firing activity.

Developmental expression of Na+ currents in mouse Purkinje neurons

As Purkinje neurons mature during postnatal development, they change from electrically quiescent to active and exhibit high frequency spontaneous action potentials. This change in electrical activity is determined by both alteration in ion channel expression and the acquisition of synaptic input. To gain a better understanding of the development the intrinsic electrical properties of these neurons, acutely isolated Purkinje neurons from mice aged postnatal day 4 (P4) to P18 were examined. This included recording action potential frequency, threshold, height and slope, and input resistance and capacitance. Changes in a number of these properties were observed, suggesting significant changes in voltage-gated Na(+) currents. Because voltage-gated Na(+) currents, including the transient, resurgent and persistent currents, are known to play important roles in generating spontaneous action potentials, the developmental changes in these currents were examined. A large increase in the density of transient current, resurgent current and persistent current was observed at times corresponding with changes in action potential properties. Interestingly, the developmental up-regulation of the persistent current and resurgent current occurred at rate which was faster than the up-regulation of the transient current. Moreover, the relative amplitudes of the persistent and resurgent currents increased in parallel, suggesting that they share a common basis. The data indicate that developmental up-regulation of Na(+) currents plays a key role in the acquisition of Purkinje neuron excitability.

Schwann Cell Engraftment Into Injured Peripheral Nerve Prevents Changes in Action Potential Properties

Peripheral nerve injury results in changes in action potential waveform, ion channel organization, and firing properties of primary afferent neurons. It has been suggested that these changes are the result of reduction in basal trophic support from skin targets. Subcutaneous injections of Fluro-Gold (FG) in the hind limb of the rat were used to identify cutaneous primary afferent neurons. Five days after FG injection, sciatic nerves were ligated and encapsulated in a silicon tube allowing neuroma formation. Green fluorescent protein (GFP)-expressing Schwann cells (SCs) were injected proximal to the cut end of the nerve. Thirteen to 22 days after injury and SC injection, the L4 and L5 dorsal root ganglia (DRG) were prepared for acute culture. Whole cell patch-clamp recordings in current clamp mode were obtained and action potential properties of medium-sized (34-45 microm) FG+ DRG neurons were characterized. In the neuroma group without cell transplantation, action potential duration and spike inflections were reduced as were the amplitude and duration of spike afterhyperpolarizations. These changes were not observed after transection by nerve crush where axons were allowed to regenerate to distal peripheral targets. In the transplantation group, GFP(+)-SCs were extensively distributed throughout the neuroma, and oriented longitudinally along axons proximal to the neuroma. Changes in action potential properties were attenuated in the GFP(+)-SC group. Thus the engrafted SC procedure ameliorated the changes in action potential waveform of cutaneous primary afferents associated with target disconnection and neuroma formation.

Effects of linoleic acid metabolites on electrical activity in adult rat ventricular myocytes

Leukotoxin (Lx), an epoxide derivative of linoleic acid, has been suggested to be a toxic mediator of multiple organ failure in burn patients and of acute respiratory distress syndrome. Lx production was recently shown during myocardial ischemia/reperfusion. However, a recent study suggested that to be toxic Lx must be metabolized to Lx-diol. In the present study, isolated adult rat ventricular myocytes were studied with the whole-cell patch-clamp technique to determine the effects of these compounds on cardiac electrical activity. Measurements of action potentials showed that neither linoleic acid nor Lx (100 μM) caused any significant changes in action potential properties. However, Lx-diol in the range of 10–100 μM produced a dose dependent increase in duration and a decrease in overshoot of the action potential. Subsequent voltage clamp experiments isolating Na current ( I Na) and transient outward K current ( I to) revealed that Lx-diol inhibited I Na and I to by about 80% at 100 μM, while linoleic acid and Lx had no effect on these currents at the same concentration. While Lx-diol produced the same inhibition of I Na and I to at 100 μM, its effects were more potent on I to with significant inhibition at 10 μM. Lx-diol also hastened the activation kinetics of I to but not I Na. The action of Lx-diol was rapid (reaching steady state in 3–5 min) and was reversible in 5–10 min following washout. Thus, Lx-diol could favor arrhythmias or cardiac arrest in intact heart and may be responsible for the cardiac problems seen in systemic inflammatory response syndrome. These results further support the suggestion that Lx is not toxic in the heart but rather must be metabolized to Lx-diol to produce toxic effects on cardiac muscle.

Development of ionic currents underlying changes in action potential waveforms in rat spinal motoneurons.

Development of ionic currents underlying changes in action potential waveforms in rat spinal motoneurons. J. Neurophysiol. 80: 3047-3061, 1998. Differentiation of the ionic mechanism underlying changes in action potential properties was investigated in spinal motoneurons of embryonic and postnatal rats using whole cell voltage- and current-clamp recordings. Relatively slow-rising, prolonged, largely Na+-dependent action potentials were recorded in embryonic motoneurons, and afterdepolarizing potentials were elicited in response to prolonged intracellular injections of depolarizing currents. Action potential amplitude, as well as its rates of rise and repolarization significantly increased, and an afterhyperpolarizing potential (AHP) became apparent immediately after birth. Concurrently, repetitive action potential firing was elicited in response to a prolonged current injection. To determine the ionic mechanism underlying these changes, the properties of voltage-gated macroscopic Na+, Ca2+, and K+ currents were examined. Fast-rising Na+ currents (INa) and slow-rising Ca2+ currents (ICa) were expressed early in embryonic development, but only INa was necessary and sufficient to trigger an action potential. INa and ICa densities significantly increased while the time to peak INa and ICa decreased after birth. The postnatal increase in INa resulted in overshooting action potential with significantly faster rate of rise than that recorded before birth. Properties of three types of outward K+ currents were examined: transient type-A current (IA), noninactivating delayed rectifier-type current (IK), and Ca2+-dependent K+ current (IK(Ca)). The twofold postnatal increase in IK and IK(Ca) densities resulted in shorter duration action potential and the generation of AHP. Relatively large IA was expressed early in neuronal development, but unlike IK and IK(Ca) its density did not increase after birth. The three types of K+ channels had opposite modulatory actions on action potential firing behavior: IK and IA increased the firing rate, whereas IK(Ca) decreased it. Our findings demonstrated that the developmental changes in action potential waveforms and the onset of repetitive firing were correlated with large increases in the densities of existing voltage-gated ion channels rather than the expression of new channel types.

Developmental changes in action potential properties of the guinea-pig myocardium.

Developmental changes in action potential properties were examined in electrically driven (1 Hz) left atria and right ventricles from foetal, neonatal and adult guinea-pig hearts, using standard micro-electrode recording techniques. In both left atria and right ventricles, the overshoot, resting potential and maximum upstroke velocity of the action potential increased progressively with age until birth, and then remained almost unchanged. Action potential duration (APD) changed markedly with age during foetal and neonatal periods. In left atria, APD at 50% repolarization initially decreased until foetal day 50, and then increased until the adult period. In right ventricles, APD initially increased until approximately foetal day 45, then decreased for 5 days following birth, thereafter it increased again. In addition, after-hyperpolarization was observed only in left atria of younger foetuses. Thus we have demonstrated that in the guinea-pig myocardium developmental changes in action potential properties occur more extensively during the foetal period than during the postnatal period.

Changes in action potential kinetics following experimental bladder outflow obstruction in the guinea pig.

The effect of experimental bladder outflow obstruction on membrane electrical activity of guinea pig detrusor smooth muscle was studied. Using an intracellular microelectrode technique, action potentials were recorded from single smooth muscle cells to determine the effect of outflow obstruction on action potential (AP) kinetics. Bladder outflow obstruction resulted in smooth muscle hypertrophy with bladder weight gain to 2.7 times control levels after 8-12 weeks' obstruction. The changes in the AP kinetics noted with obstruction-induced bladder hypertrophy were a prolongation of the AP duration and a decrease in the maximum velocity of depolarization and repolarization. The AP amplitude, after hyperpolarization and overshoot potential in addition to the resting membrane potential (RMP) did not change significantly with bladder outflow obstruction. The values of these AP parameters were not affected significantly by the application of atropine and guanethidine in smooth muscle tissue from either control or obstructed bladders. These results suggest that the active electrical properties of the detrusor smooth muscle membrane are changed significantly by obstruction-induced bladder hypertrophy. Furthermore, the results suggest that adrenergic and cholinergic neurotransmitters do not contribute to these changes in AP kinetics following obstruction. The changes in AP properties with outflow obstruction-induced bladder hypertrophy were compared with those previously reported for the hypertrophic myocardium and were discussed in relation to the known impaired contractile properties of obstructed bladder smooth muscle.

O-399 - Developmental changes in action potential properties of guinea-pig hearts

O-399 - Developmental changes in action potential properties of guinea-pig hearts

Sodium and calcium components of the action potential in a developing skeletal muscle cell line.

1. Developmental changes in action potential properties were studied in a clonal rat skeletal muscle cell line. 2. Small action potentials were evoked in mononucleate myoblasts. No spike was seen in Na-free saline. A similar spike was evoked in a medium where all NaCl was replaced by LiCl. No spike was evoked when NaCl was replaced by CsCl. 3. Action potentials overshot zero membrane potential in multinucleate myotubes. The action potential was composed of two components, an initial fast spike and a hump on the falling phase or in some cases a distinct second peak. 4. Teh overshoot of the initial fast spike decreased when the external Na concentration was decreased. 5. In saline with 10 mM-Ca the second component often formed a distinct peak following the initial fast spike. A slow regenerative potential was evoked in Na-free media with a depolarizing current pulse. 6. In saline containing BaCl-2 instead of CaCl-2 there was always a second peak, the overshoot of which changed with external Ba concentration. A slow regenerative potential was evoked in Na-free, Ba-saline. The membrane conductance at the peak of the Ba-action potential was larger than in the resting state. 7. In adult rat skeletal muscle, the shape of the action potential was not changed when Ca was replaced by Ba. No action potential was evoked in Na-free Ba-saline or Ba-saline with tetrodotoxin (3 times 10-7 M). 8. The significance of the Ca component in the developing muscle is discussed.