Analysis of cardiohemodynamic and electrophysiological effects of morphine along with its toxicokinetic profile using the halothane-anesthetized dogs.

Although morphine has been used for treatment-resistant dyspnea in end-stage heart failure patients, information on its cardiovascular safety profile remains limited. Morphine was intravenously administered to halothane-anesthetized dogs (n=4) in doses of 0.1, 1 and 10 mg/kg/10 min with 20 min of observation period. The low and middle doses attained therapeutic (0.13 µg/mL) and supratherapeutic (0.97 µg/mL) plasma concentrations, respectively. The low dose hardly altered any of the cardiovascular variables except that the QT interval was prolonged for 10-15 min after its start of infusion. The middle dose reduced the preload and afterload to the left ventricle for 5-15 min, then decreased the left ventricular contractility and mean blood pressure for 10-30 min, and finally suppressed the heart rate for 15-30 min. Moreover, the middle dose gradually but progressively prolonged the atrioventricular conduction time, QT interval/QTcV, ventricular late repolarization period and ventricular effective refractory period without altering the intraventricular conduction time, ventricular early repolarization period or terminal repolarization period. A reverse-frequency-dependent delay of ventricular repolarization was confirmed. The high dose induced cardiohemodynamic collapse mainly due to vasodilation in the initial 2 animals by 1.9 and 3.3 min after its start of infusion, respectively, which needed circulatory support to treat. The high dose was not tested further in the remaining 2 animals. Thus, intravenously administered morphine exerts a rapidly appearing vasodilator action followed by slowly developing cardiosuppressive effects. Morphine can delay the ventricular repolarization possibly through IKr inhibition in vivo, but its potential to develop torsade de pointes will be small.

The ion channel basis of pharmacological effects of amiodarone on myocardial electrophysiological properties, a comprehensive review.

Amiodarone is a benzofuran-based class III antiarrhythmic agent frequently used for the treatment of atrial and ventricular arrhythmias. The primary target of class III antiarrhythmic drugs is the cardiac human ether-a-go-go-related gene (hERG) encoded channel, KCNH2, commonly known as HERG, that conducts the rapidly activating delayed rectifier potassium current (IKr). Like other class III antiarrhythmic drugs, amiodarone exerts its physiologic effects mainly through IKr blockade, delaying the repolarization phase of the action potential and extending the effective refractory period. However, while many class III antiarrhythmics, including sotalol and dofetilide, can cause long QT syndrome (LQTS) that can progress to torsade de pointes, amiodarone displays less risk of inducing this fatal arrhythmia. This review article discusses the arrhythmogenesis in LQTS from the aspects of the development of early afterdepolarizations (EADs) associated with Ca2+ current, transmural dispersion of repolarization (TDR), as well as reverse use dependence associated with class III antiarrhythmic drugs to highlight electropharmacological effects of amiodarone on the myocardium.

Cannabidiol activates neuronal Kv7 channels.

Cannabidiol (CBD), a chemical found in the Cannabis sativa plant, is a clinically effective antiepileptic drug whose mechanism of action is unknown. Using a fluorescence-based thallium flux assay, we performed a large-scale screen and found enhancement of flux through heterologously expressed human Kv7.2/7.3 channels by CBD. Patch-clamp recordings showed that CBD acts at submicromolar concentrations to shift the voltage dependence of Kv7.2/7.3 channels in the hyperpolarizing direction, producing a dramatic enhancement of current at voltages near -50 mV. CBD enhanced native M-current in mouse superior cervical ganglion starting at concentrations of 30 nM and also enhanced M-current in rat hippocampal neurons. The potent enhancement of Kv2/7.3 channels by CBD may contribute to its effectiveness as an antiepileptic drug by reducing neuronal hyperexcitability.

Human iPSC-Cardiomyocytes as an Experimental Model to Study Epigenetic Modifiers of Electrophysiology.

The epigenetic landscape and the responses to pharmacological epigenetic regulators in each human are unique. Classes of epigenetic writers and erasers, such as histone acetyltransferases, HATs, and histone deacetylases, HDACs, control DNA acetylation/deacetylation and chromatin accessibility, thus exerting transcriptional control in a tissue- and person-specific manner. Rapid development of novel pharmacological agents in clinical testing-HDAC inhibitors (HDACi)-targets these master regulators as common means of therapeutic intervention in cancer and immune diseases. The action of these epigenetic modulators is much less explored for cardiac tissue, yet all new drugs need to be tested for cardiotoxicity. To advance our understanding of chromatin regulation in the heart, and specifically how modulation of DNA acetylation state may affect functional electrophysiological responses, human-induced pluripotent stem-cell-derived cardiomyocyte (hiPSC-CM) technology can be leveraged as a scalable, high-throughput platform with ability to provide patient-specific insights. This review covers relevant background on the known roles of HATs and HDACs in the heart, the current state of HDACi development, applications, and any adverse cardiac events; it also summarizes relevant differential gene expression data for the adult human heart vs. hiPSC-CMs along with initial transcriptional and functional results from using this new experimental platform to yield insights on epigenetic control of the heart. We focus on the multitude of methodologies and workflows needed to quantify responses to HDACis in hiPSC-CMs. This overview can help highlight the power and the limitations of hiPSC-CMs as a scalable experimental model in capturing epigenetic responses relevant to the human heart.

Electrophysiological responses to seizurogenic compounds dependent on E/I balance in human iPSC-derived cortical neural networks.

Construction of in vitro functional assay systems using human-induced pluripotent stem cells (iPSCs) as indicators for evaluating seizure liability of compounds has been anticipated. Imbalance of excitation/inhibition (E/I) inputs triggers seizure; however, the appropriate ratio of E/I neurons for evaluating seizure liability of compounds in a human iPSC-derived neural network is unknown. Here, five neural networks with varying E/I ratios (88/12, 84/16, 74/26, 58/42, and 48/52) were constructed by altering the ratios of glutamatergic (E) and GABA (I) neurons. The responsiveness of each network against six seizurogenic compounds and two GABA receptor agonists was then examined by using six representative parameters. The 52% GABA neuron network, which had the highest ratio of GABA neurons, showed the most marked response to seizurogenic compounds, however, it suggested the possibility of producing false positives. Moreover, analytical parameters were found to vary with E/I ratio and to differ for seizurogenic compounds with different mechanism of action (MoA) even at the same E/I ratio. Clustering analysis using six parameters showed the balance of 84/16, which is the closest to the biological balance, was the most suitable for detection of concentration-dependent change and classification of the MoA of seizurogenic compounds. These results suggest the importance of using a human-iPSC-derived neural network similar to the E/I balance of the living body in order to improve the prediction accuracy in the in vitro seizure liability assessment.

Functional, electrophysiology, and molecular dynamics analysis of quercetin-induced contraction of rat vascular musculature.

Quercetin, a flavonoid abundantly present in the Mediterranean diet, is considered a vasodilator despite its recognized capability to stimulate vascular CaV1.2 channel current (ICa1.2). The present study was undertaken to assess its possible vasocontractile activity. Functional and electrophysiology experiments were performed in vitro on rat aorta rings and tail artery myocytes along with an in-depth molecular modelling analysis. The CaV1.2 channel stimulator (S)-(-)-methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl) pyridine-5-carboxylate (Bay K 8644) was used as reference compound. Quercetin and Bay K 8644 caused a significant leftward shift of KCl concentration-response curve. Neither agent affected basal muscle tone, though in rings pre-treated with thapsigargin or 15 mM KCl they caused a strong, concentration-dependent contraction. Both quercetin and Bay K 8644 potentiated the response to Ca2+ in weakly depolarised rings. At high KCl concentrations, however, quercetin caused vasorelaxation. While Bay K 8644 stimulated ICa1.2, this effect being sustained with time, quercetin-induced stimulation was transient, although the molecule in solution underwent only marginal oxidation. Quercetin transient stimulation was not affected by pre-treatment with isoprenaline, sodium nitroprusside, or dephostatin; however, it converted to a sustained one in myocytes pre-incubated with Gö6976. Classical molecular dynamics simulations revealed that quercetin and Bay K 8644 formed hydrogen bonds with target sensing residues of CaV1.2 channel favouring the inactivated conformation. In conclusion, quercetin-induced stimulation of ICa1.2 promoted vasocontraction when Ca2+ buffering function of sarcoplasmic reticulum was impaired and/or smooth muscle cell membrane was moderately depolarised, as it may occur under certain pathological conditions.

Suppression of P2X3 receptor-mediated currents by the activation of α2A -adrenergic receptors in rat dorsal root ganglion neurons.

AIMS: The α2 -adrenergic receptor (α2 -AR) agonists have been shown to be effective in the treatment of various pain. For example, dexmedetomidine (DEX), a selective α2A -AR agonist, can be used for peripheral analgesia. However, it is not yet fully elucidated for the precise molecular mechanisms. P2X3 receptor is a major receptor processing nociceptive information in primary sensory neurons. Herein, we show that a functional interaction of α2A -ARs and P2X3 receptors in dorsal root ganglia (DRG) neurons could contribute to peripheral analgesia of DEX. METHODS: Electrophysiological recordings were carried out on rat DRG neurons, and nociceptive behavior was quantified in rats. RESULTS: The activation of α2A -ARs by DEX suppressed P2X3 receptor-mediated and α,ß-methylene-ATP (α,ß-meATP)-evoked inward currents in a concentration-dependent and voltage-independent manner. Pre-application of DEX shifted the α,ß-meATP concentration-response curve downwards, with a decrease of 50.43 ± 4.75% in the maximal current response of P2X3 receptors to α,ß-meATP in the presence of DEX. Suppression of α,ß-meATP-evoked currents by DEX was blocked by the α2A -AR antagonist BRL44408 and prevented by intracellular application of the Gi/o protein inhibitor pertussis toxin, the adenylate cyclase activator forskolin, and the cAMP analog 8-Br-cAMP. DEX also suppressed α,ß-meATP-evoked action potentials through α2A -ARs in rat DRG neurons. Finally, the activation of peripheral α2A -ARs by DEX had an analgesic effect on the α,ß-meATP-induced nociception. CONCLUSIONS: These results suggested that activation of α2A -ARs by DEX suppressed P2X3 receptor-mediated electrophysiological and behavioral activity via a Gi/o proteins and cAMP signaling pathway, which was a novel potential mechanism underlying analgesia of peripheral α2A -AR agonists.

Astrocytes play a critical role in mediating the effect of acute ethanol on central amygdala glutamatergic transmission.

The Central Amygdala (CeA) has been heavily implicated in many aspects of alcohol use disorder. Ethanol (EtOH) has been shown to modulate glutamatergic transmission in the lateral subdivision of the CeA, however, the exact mechanism of this modulation is still unclear. EtOH exposure is associated with increased pro-inflammatory cytokines in the CeA, and inhibition of neuroimmune cells (microglia and astrocytes) has previously been shown to reduce EtOH drinking in animal models. Since neuroimmune activation seems to be involved in many of the effects of EtOH, we hypothesized that acute EtOH exposure will increase excitatory glutamatergic transmission in the CeA via modulation of neuroimmune cells. Using ex vivo brain slice whole-cell patch clamp electrophysiology, it was found that a physiologically relevant concentration of EtOH (20 mM) significantly increased presynaptic glutamatergic transmission in the CeA. Pharmacologic and chemogenetic inhibition of astrocyte function significantly reduced the ability of EtOH to modulate CeA glutamatergic transmission with minimal impact of microglia inhibition. This finding prompted additional studies examining whether direct neuroimmune activation through lipopolysaccharide (LPS) might lead to an increase in the glutamatergic transmission in the CeA. It was found that LPS modulation of glutamatergic transmission was limited by microglia activation and required astrocyte signaling. Taken together these results support the hypothesis that acute EtOH enhances lateral CeA glutamatergic transmission through an astrocyte mediated mechanism.

Independent of differences in taste, B6N mice consume less alcohol than genetically similar B6J mice, and exhibit opposite polarity modulation of tonic GABAAR currents by alcohol.

Genetic differences in cerebellar sensitivity to alcohol (EtOH) influence EtOH consumption phenotype in animal models and contribute to risk for developing an alcohol use disorder in humans. We previously determined that EtOH enhances cerebellar granule cell (GC) tonic GABAAR currents in low EtOH consuming rodent genotypes, but suppresses it in high EtOH consuming rodent genotypes. Moreover, pharmacologically counteracting EtOH suppression of GC tonic GABAAR currents reduces EtOH consumption in high alcohol consuming C57BL/6J (B6J) mice, suggesting a causative role. In the low EtOH consuming rodent models tested to date, EtOH enhancement of GC tonic GABAAR currents is mediated by inhibition of neuronal nitric oxide synthase (nNOS) which drives increased vesicular GABA release onto GCs and a consequent enhancement of tonic GABAAR currents. Consequently, genetic variation in nNOS expression across rodent genotypes is a key determinant of whether EtOH enhances or suppresses tonic GABAAR currents, and thus EtOH consumption. We used behavioral, electrophysiological, and immunocytochemical techniques to further explore the relationship between EtOH consumption and GC GABAAR current responses in C57BL/6N (B6N) mice. B6N mice consume significantly less EtOH and achieve significantly lower blood EtOH concentrations than B6J mice, an outcome not mediated by differences in taste. In voltage-clamped GCs, EtOH enhanced the GC tonic current in B6N mice but suppressed it in B6J mice. Immunohistochemical and electrophysiological studies revealed significantly higher nNOS expression and function in the GC layer of B6N mice compared to B6Js. Collectively, our data demonstrate that despite being genetically similar, B6N mice consume significantly less EtOH than B6J mice, a behavioral difference paralleled by increased cerebellar nNOS expression and opposite EtOH action on GC tonic GABAAR currents in each genotype.

Simultaneous analyses of hemodynamic and electrophysiological effects of oseltamivir along with its pharmacokinetic profile using the canine paroxysmal atrial fibrillation model.

Since information of antiviral drug oseltamivir on the anti-atrial fibrillation (AF) property is still limited, we assessed it using the canine paroxysmal AF model. Oseltamivir in doses of 3 and 30 mg/kg/10 min was intravenously infused to the isoflurane-anesthetized, chronic atrioventricular block dogs (n = 6) with monitoring hemodynamic and electrophysiological variables, in which AF was induced by 10 s of burst pacing on atrial septum. Oseltamivir decreased AF incidence and AF duration, and prolonged AF cycle length in a dose-dependent manner. The low and high doses attained the peak plasma drug concentrations of 9.7 and 96.5 µg/mL, which were approximately 100 and 1000 times greater than those observed in human clinical cases, respectively. The low dose of oseltamivir decreased mean blood pressure without altering sinoatrial or idioventricular rate, whereas its high dose reduced each of them. Oseltamivir delayed inter-atrial conduction in dose- and frequency-dependent manners, whereas it prolonged atrial effective refractory period in dose-dependent but frequency-independent manners. The high dose prolonged ventricular effective refractory period, which was not detected with the low dose. These findings can be used for repurposing oseltamivir as an anti-AF drug candidate.

Suppression of ASIC activity by the activation of A1 adenosine receptors in rat primary sensory neurons.

Peripheral A1 adenosine receptor signaling has been shown to have analgesic effects in a variety of pain conditions. However, it is not yet fully elucidated for the precise molecular mechanisms. Acid sensing ion channels (ASICs) are expressed predominantly in nociceptive sensory neurons responding to protons. Given that both A1 adenosine receptors and ASICs are present in dorsal root ganglia (DRG) neurons, we therefore investigated whether there was a cross-talk between the two types of receptors. Herein, electrophysiological recordings showed that the A1 adenosine receptor agonist N6-cyclopentyladenosine (CPA) suppressed acid-induced currents and action potentials, which were mediated by ASICs, in rat DRG neurons. CPA inhibited the maximum response to protons, as shown a downward shift of concentration-response curve for protons. The CPA-induced suppression of ASIC currents was blocked by the A1 adenosine receptor antagonist KW-3902 and also prevented by intracellular application of the Gi/o-protein inhibitor pertussis toxin, the adenylate cyclase activator forskolin, and the cAMP analog 8-Br-cAMP. Finally, intraplantar pretreatment of CPA dose-dependently relieved acid-induced nociceptive responses in rats through peripheral A1 adenosine receptors. These results suggested that CPA suppressed ASICs via A1 adenosine receptors and intracellular Gi/o-proteins and cAMP signaling cascades in rat DRG neurons, which was a novel potential mechanism underlying analgesia of peripheral A1 adenosine receptors.

Upregulation of the JAK-STAT pathway promotes maturation of human embryonic stem cell-derived cardiomyocytes.

The immature characteristics and metabolic phenotypes of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) restrict their applications for disease modeling, drug discovery, and cell-based therapy. Leveraging on the metabolic shifts from glycolysis to fatty acid oxidation as CMs mature, a human hexokinase1-GFP metabolic reporter cell line (H7 HK1-GFP) was generated to facilitate the isolation of fetal or more matured hPSC-CMs. RNA sequencing of fetal versus more matured CMs uncovered a potential role of interferon-signaling pathway in regulating CM maturation. Indeed, IFN-γ-treated CMs resulted in an upregulation of the JAK-STAT pathway, which was found to be associated with increased expression of CM maturation genes, shift from MYH6 to MYH7 expression, and improved sarcomeric structure. Functionally, IFN-γ-treated CMs exhibited a more matured electrophysiological profile, such as increased calcium dynamics and action potential upstroke velocity, demonstrated through calcium imaging and MEA. Expectedly, the functional improvements were nullified with a JAK-STAT inhibitor, ruxolitinib.

A mathematical model of hiPSC cardiomyocytes electromechanics.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are becoming instrumental in cardiac research, human-based cell level cardiotoxicity tests, and developing patient-specific care. As one of the principal functional readouts is contractility, we propose a novel electromechanical hiPSC-CM computational model named the hiPSC-CM-CE. This model comprises a reparametrized version of contractile element (CE) by Rice et al., 2008, with a new passive force formulation, integrated into a hiPSC-CM electrophysiology formalism by Paci et al. in 2020. Our simulated results were validated against in vitro data reported for hiPSC-CMs at matching conditions from different labs. Specifically, key action potential (AP) and calcium transient (CaT) biomarkers simulated by the hiPSC-CM-CE model were within the experimental ranges. On the mechanical side, simulated cell shortening, contraction-relaxation kinetic indices (RT50 and RT25 ), and the amplitude of tension fell within the experimental intervals. Markedly, as an inter-scale analysis, correct classification of the inotropic effects due to non-cardiomyocytes in hiPSC-CM tissues was predicted on account of the passive force expression introduced to the CE. Finally, the physiological inotropic effects caused by Verapamil and Bay-K 8644 and the aftercontractions due to the early afterdepolarizations (EADs) were simulated and validated against experimental data. In the future, the presented model can be readily expanded to take in pharmacological trials and genetic mutations, such as those involved in hypertrophic cardiomyopathy, and study arrhythmia trigger mechanisms.

Clemastine improves electrophysiologic and histomorphometric changes through promoting myelin repair in a murine model of compression neuropathy.

Compression neuropathies are common and debilitating conditions that result in variable functional recovery after surgical decompression. Recent drug repurposing studies have verified that clemastine promotes functional recovery through enhancement of myelin repair in demyelinating disease. We investigated the utility of clemastine as a treatment for compression neuropathy using a validated murine model of compression neuropathy encircling the compression tube around the sciatic nerve. Mice received PBS or clemastine solution for 6 weeks of compression phase. Mice taken surgical decompression received PBS or clemastine solution for 2 weeks of decompression phase. Electrodiagnostic, histomorphometric, and Western immunoblotting analyses were performed to verify the effects of clemastine. During the compression phase, mice treated with clemastine had significantly decreased latency and increased amplitude compared to untreated mice that received PBS. Histomorphometric analyses revealed that mice treated with clemastine had significantly higher proportions of myelinated axons, thicker myelin, and a lower G-ratio. The expression levels of myelin proteins, including myelin protein zero and myelin associated glycoprotein, were higher in mice treated with clemastine. However, the electrophysiologic and histomorphometric improvements were observed regardless of clemastine treatment in mice taken surgical decompression. Mice treated with clemastine during compression of the sciatic nerve demonstrated that clemastine treatment attenuated electrophysiologic and histomorphometric changes caused by compression through promoting myelin repair.

BDNF shifts excitatory-inhibitory balance in the paraventricular nucleus of the hypothalamus to elevate blood pressure.

Presympathetic neurons in the paraventricular nucleus of the hypothalamus (PVN) play a key role in cardiovascular regulation. We have previously shown that brain-derived neurotrophic factor (BDNF), acting in the PVN, increases sympathetic activity and blood pressure and serves as a key regulator of stress-induced hypertensive responses. BDNF is known to alter glutamatergic and GABA-ergic signaling broadly in the central nervous system, but whether BDNF has similar actions in the PVN remains to be investigated. Here, we tested the hypothesis that increased BDNF expression in the PVN elevates blood pressure by enhancing N-methyl-d-aspartate (NMDA) receptor (NMDAR)- and inhibiting GABAA receptor (GABAAR)-mediated signaling. Sprague-Dawley rats received bilateral PVN injections of AAV2 viral vectors expressing green fluorescent protein (GFP) or BDNF. Three weeks later, cardiovascular responses to PVN injections of NMDAR and GABAAR agonists and antagonists were recorded under α-chloralose-urethane anesthesia. In addition, expressions of excitatory and inhibitory signaling components in the PVN were assessed using immunofluorescence. Our results showed that NMDAR inhibition led to a greater decrease in blood pressure in the BDNF vs. GFP group, while GABAAR inhibition led to greater increases in blood pressure in the GFP group compared to BDNF. Conversely, GABAAR activation decreased blood pressure significantly more in GFP vs. BDNF rats. In addition, immunoreactivity of NMDAR1 was upregulated, while GABAAR-α1 and K+/Cl- cotransporter 2 were downregulated by BDNF overexpression in the PVN. In summary, our findings indicate that hypertensive actions of BDNF within the PVN are mediated, at least in part, by augmented NMDAR and reduced GABAAR signaling.NEW & NOTEWORTHY We have shown that BDNF, acting in the PVN, elevates blood pressure in part by augmenting NMDA receptor-mediated excitatory input and by diminishing GABAA receptor-mediated inhibitory input to PVN neurons. In addition, we demonstrate that elevated BDNF expression in the PVN upregulates NMDA receptor immunoreactivity and downregulates GABAA receptor as well as KCC2 transporter immunoreactivity.

Electrophysiological engineering of heart-derived cells with calcium-dependent potassium channels improves cell therapy efficacy for cardioprotection.

We have shown that calcium-activated potassium (KCa)-channels regulate fundamental progenitor-cell functions, including proliferation, but their contribution to cell-therapy effectiveness is unknown. Here, we test the participation of KCa-channels in human heart explant-derived cell (EDC) physiology and therapeutic potential. TRAM34-sensitive KCa3.1-channels, encoded by the KCNN4 gene, are exclusively expressed in therapeutically bioactive EDC subfractions and maintain a strongly polarized resting potential; whereas therapeutically inert EDCs lack KCa3.1 channels and exhibit depolarized resting potentials. Somatic gene transfer of KCNN4 results in membrane hyperpolarization and increases intracellular [Ca2+], which boosts cell-proliferation and the production of pro-healing cytokines/nanoparticles. Intramyocardial injection of EDCs after KCNN4-gene overexpression markedly increases the salutary effects of EDCs on cardiac function, viable myocardium and peri-infarct neovascularization in a well-established murine model of ischemic cardiomyopathy. Thus, electrophysiological engineering provides a potentially valuable strategy to improve the therapeutic value of progenitor cells for cardioprotection and possibly other indications.

Circadian rhythm influences naloxone induced morphine withdrawal and neuronal activity of lateral paragigantocellularis nucleus.

Investigations have shown that the circadian rhythm can affect the mechanisms associated with drug dependence. In this regard, we sought to assess the negative consequence of morphine withdrawal syndrome on conditioned place aversion (CPA) and lateral paragigantocellularis (LPGi) neuronal activity in morphine-dependent rats during light (8:00-12:00) and dark (20:00-24:00) cycles. Male Wistar rats (250-300 g) were received 10 mg/kg morphine or its vehicle (Saline, 2 mL/kg/12 h, s.c.) in 13 consecutive days for behavioral assessment tests. Then, naloxone-induced conditioned place aversion and physical signs of withdrawal syndrome were evaluated during light and dark cycles. In contrast to the behavioral part, we performed in vivo extracellular single-unit recording for investigating the neural response of LPGi to naloxone in morphine-dependent rats on day 10 of morphine/saline exposure. Results showed that naloxone induced conditioned place aversion in both light and dark cycles, but the CPA score during the light cycle was larger. Moreover, the intensity of physical signs of morphine withdrawal syndrome was more severe during the light cycle (rest phase) compare to the dark one. In electrophysiological experiments, results indicated that naloxone evoked both excitatory and inhibitory responses in LPGi neurons and the incremental effect of naloxone on LPGi activity was stronger in the light cycle. Also, the neurons with the excitatory response exhibited higher baseline activity in the dark cycle, but the neurons with the inhibitory response showed higher baseline activity in the light cycle. Interestingly, the baseline firing rate of neurons recorded in the light cycle was significantly different in response (excitatory/inhibitory) -dependent manner. We concluded that naloxone-induced changes in LPGi cellular activity and behaviors of morphine-dependent rats can be affected by circadian rhythm and the internal clock.

Bidirectional flow of the funny current (If) during the pacemaking cycle in murine sinoatrial node myocytes.

Sinoatrial node myocytes (SAMs) act as cardiac pacemaker cells by firing spontaneous action potentials (APs) that initiate each heartbeat. The funny current (If) is critical for the generation of these spontaneous APs; however, its precise role during the pacemaking cycle remains unresolved. Here, we used the AP-clamp technique to quantify If during the cardiac cycle in mouse SAMs. We found that If is persistently active throughout the sinoatrial AP, with surprisingly little voltage-dependent gating. As a consequence, it carries both inward and outward current around its reversal potential of -30 mV. Despite operating at only 2 to 5% of its maximal conductance, If carries a substantial fraction of both depolarizing and repolarizing net charge movement during the firing cycle. We also show that ß-adrenergic receptor stimulation increases the percentage of net depolarizing charge moved by If, consistent with a contribution of If to the fight-or-flight increase in heart rate. These properties were confirmed by heterologously expressed HCN4 channels and by mathematical models of If Modeling further suggested that the slow rates of activation and deactivation of the HCN4 isoform underlie the persistent activity of If during the sinoatrial AP. These results establish a new conceptual framework for the role of If in pacemaking, in which it operates at a very small fraction of maximal activation but nevertheless drives membrane potential oscillations in SAMs by providing substantial driving force in both inward and outward directions.

Electrophysiological predictors of response to subcutaneous immunoglobulin therapy in chronic inflammatory demyelinating polyneuropathy.

OBJECTIVE: To assess axonal function prior to subcutaneous immunoglobulin (SCIG) therapy or placebo in relation to relapse in chronic inflammatory demyelinating polyneuropathy (CIDP) to determine whether axonal damage can predict therapy response. METHODS: Relapse rates in patients from the Polyneuropathy and Treatment with Hizentra (PATH) study, where patients were treated with placebo or SCIG (IgPro20), were analyzed by baseline (post-intravenous immunoglobulin stabilization) axonal damage (≤1 mV peroneal compound muscle action potential) status. RESULTS: In patients with non-axonal damage, relapses were significantly higher with placebo (73.0%) than IgPro20 (0.2 g/kg: 39.1%, 0.4 g/kg: 19.2%). In patients with axonal damage, IgPro20 had no effect on relapse (placebo: 25.0%, IgPro20: 0.2 g/kg: 30.0%, 0.4 g/kg: 19.4%). Patients with axonal damage relapsed significantly less on placebo versus non-axonal damage, but they also demonstrated higher baseline disability. CONCLUSION: Axonal damage may correspond to relapse upon treatment withdrawal; patients with axonal damage relapse less, possibly reflecting poor response to immunoglobulin therapy, while non-axonal damage patients may experience more relapse, perhaps indicating better treatment response. SIGNIFICANCE: In CIDP patients with axonal loss, immunoglobulin therapy may not be as effective. Assessing axonal damage could help guide therapy, with immunoglobulins ideally used before substantial axonal damage arises.

Spike Activity in the Ventromedial Nucleus of Rat Hypothalamus during Aging.

Spike activity of neurons in the ventromedial nucleus (VMN) of the hypothalamus in adult (6-8 months) and aged (2 years) male rats was studied by the in vivo extracellular method using stereotaxic insertion of microelectrodes. In all animals, firing frequency of most VMN neurons increased in response to glucose administration. However, in aged rats, the mean baseline and glucose-induced spike frequencies of VMN neurons were lower than in adult animals. These results support the hypothesis that aging is associated with a decrease in the functional activity of hypothalamic neurons.