Spontaneous Action Potential Rate Research Articles

A Mathematical Model of Spontaneous Action Potential Based on Stochastics Synaptic Noise Dynamics in Non-Neural Cells

We developed a mathematical model to simulate the dynamics of background synaptic noise in non-neuronal cells. By employing the stochastic Ornstein–Uhlenbeck process, we represented excitatory synaptic conductance and integrated it into a whole-cell model to generate spontaneous and evoke cellular electrical activities. This single-cell model encompasses numerous biophysically detailed ion channels, depicted by a set of ordinary differential equations in Hodgkin–Huxley and Markov formalisms. Consequently, this approach effectively induced irregular spontaneous depolarizations (SDs) and spontaneous action potentials (sAPs), resembling electrical activity observed in vitro. The input resistance decreased significantly, while the firing rate of spontaneous action potentials increased. Moreover, alterations in the ability to reach the action potential threshold were observed. Background synaptic activity can modify the input/output characteristics of non-neuronal excitatory cells. Hence, suppressing these baseline activities could aid in identifying new pharmaceutical targets for various clinical diseases.

Characterization and mu opioid receptor sensitivity of neuropeptide Y interneurons in the mouse nucleus accumbens

Inhibitory interneurons represent less than 5% of neurons within the nucleus accumbens, but are critical for proper microcircuit function within this brain region. In the dorsal striatum, neuropeptide Y is expressed by two interneuron subtypes (low-threshold spiking interneurons and neurogliaform interneurons) that exhibit mu opioid receptor sensitivity in other brain regions. However, few studies have assessed the molecular and physiological properties of neuropeptide Y interneurons within the nucleus accumbens. We used a transgenic reporter mouse to identify and characterize neuropeptide Y interneurons in acute nucleus accumbens brain slices. Nearly all cells exhibited electrophysiological properties of low-threshold spiking interneurons, with almost no neurogliaform interneurons observed among neuropeptide Y interneurons. We corroborated this pattern using fluorescent in situ hybridization, and also identified a high level of mu opioid receptor expression by low-threshold spiking interneurons, which led us to examine the functional consequences of mu opioid receptor activation in these cells using electrophysiology. Mu opioid receptor activation caused a reduction in the rate of spontaneous action potentials in low-threshold spiking interneurons, as well as a decrease in optogenetically-evoked GABA release onto medium spiny neurons. The latter effect was more robust in female versus male mice, and when the postsynaptic medium spiny neuron expressed the Drd1 dopamine receptor. This work is the first to examine the physiological properties of neuropeptide Y interneurons in the nucleus accumbens, and show they may be an important target for mu opioid receptor modulation by endogenous and exogenous opioids.

Effectiveness in the inhibition of dapagliflozin and canagliflozin on M-type K+ current and α-methylglucoside-induced current in pituitary tumor (GH3) and pheochromocytoma PC12 cells

Dapagliflozin (DAPA) or canagliflozin (CANA), Na+-dependent glucose co-transporter type 2 (SGLT2) inhibitors, were used for treatment of type II diabetes mellitus. Addition of DAPA or CANA suppressed M-type K+ current (IK(M)) in pituitary tumor (GH3) and pheochromocytoma PC12 cells. The IC50 value for DAPA- or CANA-mediated inhibition of IK(M) in GH3 cells was 0.11 or 0.42 μM, respectively. The presence of DAPA (0.1 μM) shifted the steady-state activation of IK(M) to less depolarized potential without changing the gating charge of the current. During high-frequency depolarizing pulses, IK(M) magnitude was reduced by DAPA; however, DAPA-induced block of IK(M) remained effective. The amplitude of neither erg-mediated K+ current nor hyperpolarization-activated cation current in GH3 cells was modified in the presence of 1 μM DAPA. Alternatively, addition of DAPA, CANA, phlorizin or chlorotoxin effectively suppressed α-methylglucoside-(αMG-) induced current (IαMG) in GH3 cells, albeit inability of tefluthrin (activator of INa) to suppress this current. DAPA shifted the charge-voltage relation of presteady-state IαMG in a rightward and downward direction with no change in the gating charge of the IαMG. Under current-clamp recordings, subsequent additions of DAPA, but still in the continued presence of αMG, increased the firing rate of spontaneous action potentials stimulated by αMG. Our results suggested that activity of SGLT was expressed functionally in GH3 and PC12 cells. Therefore, inhibitory actions of DAPA or CANA on the amplitude and gating of IK(M) might provide a yet unidentified mechanism through which the SGLT1 or SGLT2 activity were attenuated in unclamped cells occurring in vivo.

Inhibitory Effects of Cyclopiazonic Acid on the Pacemaker Current in Sinoatrial Nodal Cells

Objective: The spontaneous action potential of isolated sinoatrial node (SAN) cells is regulated by a coupled-clock system of two clocks: the calcium clock and membrane clock. However, it remains unclear whether calcium clock inhibitors have a direct effect on the membrane clock. The purpose of this study was to investigate the direct effect of cyclopiazonic acid (CPA), a selective calcium clock inhibitor, on the function of the membrane clock of SAN cells. Methods: at SAN cells were isolated by trypsinization and identified based on morphology and electrophysiology. If and HCN currents were recorded via patch clamp technique. The expression of the HCN channel protein was determined by Western blotting analysis. Results: The diastolic depolarization rate of spontaneous action potentials and the current densities of If were reduced by exposure to 10 μM CPA. The inhibitory effect of CPA was concentration-dependent with an IC50 value of 16.3 μM and a Hill coefficient of 0.98. The effect of CPA on If current was also time-dependent, and the If current amplitude was partially restored after washout. Furthermore, the steady-state activation curve of the If current was shifted to a negative potential, indicating that channel activation slowed down. Finally, the protein expression of HCN4 in HEK293 cells was markedly downregulated by CPA. Conclusions: These results indicate that the direct inhibition effect of CPA on the If current in SAN cells is both concentration- and time-dependent. The underlying mechanisms may involve slowing down steady-state activation and the downregulation of pacemaker channel protein expression.

Bipolar Disorder That Responds to Lithium May Be Distinct Illness, Says Researcher

Back to table of contents Previous article Next article Clinical and Research NewsFull AccessBipolar Disorder That Responds to Lithium May Be Distinct Illness, Says ResearcherAaron LevinAaron LevinSearch for more papers by this authorPublished Online:20 Jul 2017https://doi.org/10.1176/appi.pn.2017.7b4AbstractSearching the genome to identify super responders to lithium may sharpen focus of treatment and shorten the time to diagnosis and stabilization for those with bipolar illness.Increasing evidence suggests that bipolar disorder that improves with the use of lithium is a genetically distinct form of the illness, said John Kelsoe, M.D., at APA’s 2017 Annual Meeting in San Diego in May.Mrazek Award winner John Kelsoe, M.D. (second from right), is photographed with (from left) James Kennedy, M.D., who chaired Kelsoe’s lecture, and family members of the psychiatrist after whom the award is named: daughter Alissa Mrazek, Ph.D., wife Patricia Mrazek, Ph.D., and son Michael Mrazek, Ph.D.Ellen DallagerKelsoe made his remarks after being presented with the David A. Mrazek, M.D., Memorial Award, which recognizes an outstanding clinician in the field of pharmacogenomics.“Bipolar disorder is about 70 to 80 percent genetic, and genomewide association studies have discovered at least 19 to 30 genes so far,” said Kelsoe, a professor of psychiatry and director of the Laboratory for Psychiatric Genomics at the University of California, San Diego. The prevalence rate in the general population is about 1 percent, but it is 7 to 10 percent among relatives of patients. People carrying some of the alleles might be hyperthymic and more evolutionarily fit, helping to preserve the genes in the population. Bipolar illness arises when that genetic load gets too high.“If we look for genes that control lithium response, we might find some that are associated with both lithium response and bipolar disorder,” he said. “I propose that these are genes that predispose to a form of bipolar disorder that causes dysfunction in pathways modulated by lithium and define mechanistically separate forms of the illness.”If such a distinct form of the illness can be detected, thus predicting treatment response, then patients could be diagnosed and treated sooner, he said. On average now, a person with bipolar disorder goes seven years without an accurate diagnosis. In that period, the individual is likely to see at least three physicians and is often misdiagnosed and may be put on drugs that don’t work or may make the condition worse.Running full-blown genomewide association tests on patients is expensive and probably not productive at this point. However, using pluripotent stem cells could identify candidate genes that indicate lithium responders, a new technology Kelsoe called “the most revolutionary thing in psychiatry in 100 years.” It is now being explored by a number of researchers.Neurons derived from patients’ stem cells grown in the lab betray their origins, he said. “Bipolar neurons are like bipolar patients—very busy.”Those cells exhibit three times the rate of spontaneous action potentials compared with cells from healthy individuals. Adding lithium to stem cells from lithium-responding patients rescues them from the high firing rate. “We recapitulated in the dish the clinical response we saw in the patients, but there was little effect on cells from nonresponders,” he said.Kelsoe suggested that bipolar patients may show decreased function of GABA receptors and increased function of voltage-gated calcium channels, a combination that alters the action of pyramidal cells by “inhibiting the inhibitors.”“This is probably a defect in the channel that lets ions into the cell,” he said. “What that means is unclear, but that cellular phenotype of hyperexcitability gives us something to look for.”Once it took eight weeks to grow stem cells; now it can be done in two days. That time may decrease to the point that predicting drug response could take place while the patient waits in the clinic.“We’d like to identify the diagnosis early on based on DNA testing that points to a specific treatment and gets the patient better in a couple of months versus a couple of years,” he said. ■“Genetic Variants Associated With Response to Lithium Treatment In Bipolar Disorder: A Genome-Wide Association Study” can be accessed here. ISSUES NewArchived

Multiple ion channel block by the cation channel inhibitor SKF-96365 in myocytes from the rabbit atrioventricular node

The atrioventricular node (AVN) of the cardiac conduction system coordinates atrial–ventricular excitation and can act as a subsidiary pacemaker. Recent evidence suggests that an inward background sodium current, I B,Na, carried by nonselective cation channels (NSCCs), contributes to AVN cell pacemaking. The study of the physiological contribution of I B,Na has been hampered, however, by a lack of selective pharmacological antagonists. This study investigated effects of the NSCC inhibitor SKF‐96365 on spontaneous activity, I B,Na, and other ionic currents in AVN cells isolated from the rabbit. Whole‐cell patch‐clamp recordings of action potentials (APs) and ionic currents were made at 35–37°C. A concentration of 10 μmol/L SKF‐96365 slowed spontaneous action potential rate by 13.9 ± 5.3% (n = 8) and slope of the diastolic depolarization from 158.1 ± 30.5 to 86.8 ± 30.5 mV sec−1 (P < 0.01; n = 8). Action potential upstroke velocity and maximum diastolic potential were also reduced. Under I B,Na‐selective conditions, 10 μmol/L SKF‐96365 inhibited I B,Na at −50 mV by 36.1 ± 6.8% (n = 8); however, effects on additional channel currents were also observed. Thus, the peak l‐type calcium current (I Ca,L) at +10 mV was inhibited by 38.6 ± 8.1% (n = 8), while the rapid delayed rectifier current, I Kr, tails at −40 mV following depolarization to +20 mV were inhibited by 55.6 ± 4.6% (n = 8). The hyperpolarization‐activated current, I f, was unaffected by SKF‐96365. Collectively, these results indicate that SKF‐96365 exerts a moderate inhibitory effect on I B,Na and slows AVN cell pacemaking. However, additional effects of the compound on I Ca,L and I Kr confound the use of SKF‐96365 to dissect out selectively the physiological role of I B,Na in the AVN.

Direct conversion of human fibroblasts to induced serotonergic neurons.

Serotonergic (5HT) neurons exert diverse and widespread functions in the brain. Dysfunction of the serotonergic system gives rise to a variety of mental illnesses including depression, anxiety, obsessive compulsive disorder, autism and eating disorders. Here we show that human primary fibroblasts were directly converted to induced serotonergic (i5HT) neurons by the expression of Ascl1, Foxa2, Lmx1b and FEV. The transdifferentiation was enhanced by p53 knockdown and appropriate culture conditions including hypoxia. The i5HT neurons expressed markers for mature serotonergic neurons, had Ca2+-dependent 5HT release and selective 5HT uptake, exhibited spontaneous action potentials and spontaneous excitatory postsynaptic currents. Application of serotonin significantly increased the firing rate of spontaneous action potentials, demonstrating the functional utility of i5HT neurons for studying serotonergic neurotransmission. The availability of human i5HT neurons will be very useful for research and drug discovery on many serotonin-related mental disorders.

Effect of Exogenous Extracellular Nicotinamide Adenine Dinucleotide (NAD⁺) on Bioelectric Activity of the Pacemaker and Conduction System of the Heart.

In rat sinoatrial node, NAD(+) (10 μM) reduced the rate of spontaneous action potentials, duration of action potentials, and the velocity of slow diastolic depolarization, but the rate of action potential front propagation increases. In passed rabbit Purkinje fibers, NAD(+) (10 μM) reduced the duration of action potentials. Under conditions of spontaneous activity of Purkinje fibers, NAD(+) reduced the fi ring rate and the rate of slow diastolic depolarization. The effects of extracellular NAD(+) on bioelectric activity of the pacemaker (sinoatrial node) and conduction system of the heart (Purkinje fibers) are probably related to activation of P1 and P2 purinoceptors.

Ionic mechanisms underlying the negative chronotropic action of propofol on sinoatrial node automaticity in guinea pig heart.

Propofol is a widely used intravenous anaesthetic agent, but has undesirable cardiac side effects, including bradyarrhythmia and its severe form asystole. This study examined the ionic and cellular mechanisms underlying propofol-induced bradycardia. Sinoatrial node cells, isolated from guinea pig hearts, were current- and voltage-clamped to record action potentials and major ionic currents involved in their spontaneous activity, such as the hyperpolarization-activated cation current (If ), T-type and L-type Ca(2+) currents (ICa,T and ICa,L , respectively) and the rapidly and slowly activating delayed rectifier K(+) currents (IKr and IK s , respectively). ECGs were recorded from Langendorff-perfused, isolated guinea pig hearts. Propofol (≥5 μM) reversibly decreased the firing rate of spontaneous action potentials and their diastolic depolarization rate. Propofol impaired If activation by shifting the voltage-dependent activation to more hyperpolarized potentials (≥1 μM), slowing the activation kinetics (≥3 μM) and decreasing the maximal conductance (≥10 μM). Propofol decreased ICa,T (≥3 μM) and ICa,L (≥1 μM). Propofol suppressed IKs (≥3 μM), but had a minimal effect on IKr . Furthermore, propofol (≥5 μM) decreased heart rates in Langendorff-perfused hearts. The sinoatrial node cell model reasonably well reproduced the negative chronotropic action of propofol. Micromolar concentrations of propofol suppressed the slow diastolic depolarization and firing rate of sinoatrial node action potentials by impairing If activation and reducing ICa,T , ICa,L and IKs . These observations suggest that the direct inhibitory effect of propofol on sinoatrial node automaticity, mediated via multiple channel inhibition, underlies the propofol-induced bradycardia observed in clinical settings.

Increased firing frequency of spontaneous action potentials in cerebellar Purkinje neurons of db/db mice results from altered auto-rhythmicity and diminished GABAergic tonic inhibition

The hormone leptin, by binding to hypothalamic receptors, suppresses food intake and decreases body adiposity. Leptin receptors are also widely expressed in extra-hypothalamic areas such as hippocampus, amygdala and cerebellum, where leptin modulates synaptic transmission. Here we show that a defective leptin receptor affects the electrophysiological properties of cerebellar Purkinje neurons (PNs). PNs from (db/db) mice recorded in cerebellar slices display a higher firing rate of spontaneous action potentials than PNs from wild type (WT) mice. Blockade of GABAergic tonic inhibition with bicuculline in WT mice changes the firing pattern from continuous, uninterrupted spiking into bursting firing, but bicuculline does not produce these alterations in db/db neurons, suggesting that they receive a weaker GABAergic inhibitory input. Our results also show that the intrinsic firing properties (auto-rhythmicity) of WT and db/db PNs are different. Tonic firing of PNs, the only efferent output from the cerebellar cortex, is a persistent signal to downstream cerebellar targets. The significance of leptin modulation of PNs spontaneous firing is not known. Also, it is not clear if the increased excitability of cerebellar PNs in db/db mice results from hyperglycemia or from the lack of leptin signaling, since both conditions coexist in the db/db strain.

Inhibitory effects of sevoflurane on pacemaking activity of sinoatrial node cells in guinea-pig heart

The volatile anaesthetic sevoflurane affects heart rate in clinical settings. The present study investigated the effect of sevoflurane on sinoatrial (SA) node automaticity and its underlying ionic mechanisms. Spontaneous action potentials and four ionic currents fundamental for pacemaking, namely, the hyperpolarization-activated cation current (I(f) ), T-type and L-type Ca²⁺ currents (I(Ca,T) and I(Ca,L) , respectively), and slowly activating delayed rectifier K⁺ current (I(Ks) ), were recorded in isolated guinea-pig SA node cells using perforated and conventional whole-cell patch-clamp techniques. Heart rate in guinea-pigs was recorded ex vivo in Langendorff mode and in vivo during sevoflurane inhalation. In isolated SA node cells, sevoflurane (0.12-0.71 mM) reduced the firing rate of spontaneous action potentials and its electrical basis, diastolic depolarization rate, in a qualitatively similar concentration-dependent manner. Sevoflurane (0.44 mM) reduced spontaneous firing rate by approximately 25% and decreased I(f) , I(Ca,T) , I(Ca,L) and I(Ks) by 14.4, 31.3, 30.3 and 37.1%, respectively, without significantly affecting voltage dependence of current activation. The negative chronotropic effect of sevoflurane was partly reproduced by a computer simulation of SA node cell electrophysiology. Sevoflurane reduced heart rate in Langendorff-perfused hearts, but not in vivo during sevoflurane inhalation in guinea-pigs. Sevoflurane at clinically relevant concentrations slowed diastolic depolarization and thereby reduced pacemaking activity in SA node cells, at least partly due to its inhibitory effect on I(f) , I(Ca,T) and I(Ca,L) . These findings provide an important electrophysiological basis of alterations in heart rate during sevoflurane anaesthesia in clinical settings.

Excitation of locus coeruleus noradrenergic neurons by thyrotropin‐releasing hormone

Locus coeruleus (LC) noradrenergic neurons are implicated in a variety of functions including the regulation of vigilance and the modulation of sensory processing. Thyrotropin-releasing hormone (TRH) is an endogenous neuropeptide that induces a variety of behavioural changes including arousal and antinociception. In the present study, we explored whether the activity of LC noradrenergic neurons is modulated by TRH. Using current-clamp recording from isolated rat LC neurons, we found that TRH increased the firing rate of spontaneous action potentials. The TRH action was mimicked by TRH analogues including taltirelin and TRH-gly. In voltage-clamp recording at a holding potential of 50 mV, TRH produced an inward current associated with a decrease in the membrane K+ conductance. This current was inhibited by the TRH receptor antagonist chlordiazepoxide. Following inhibition of the pH-sensitive K+ conductance by extracellular acidification, the TRH response was fully inhibited. The TRH-induced current was also inhibited by the phospholipase C (PLC) inhibitor U-73122, but not by the protein kinase C inhibitor chelerythrine nor by chelation of intracellular Ca2+ by BAPTA. The recovery from the facilitatory action of TRH on the spike frequency was markedly inhibited by a high concentration of wortmannin. These results suggest that TRH activates LC noradrenergic neurons by decreasing an acid-sensitive K+ conductance via PLC-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate. The present findings demonstrate that TRH activates LC neurons and characterize the underlying signalling mechanisms. The action of TRH on LC neurons may influence a variety of CNS functions related to the noradrenergic system which include arousal and analgesia.

Acidosis inhibits spontaneous activity and membrane currents in myocytes isolated from the rabbit atrioventricular node

Recent evidence from intact hearts suggests that the function of cardiac nodal tissue may be particularly susceptible to acidosis. Little is currently known, however, about the effects of acidosis on the cellular electrophysiology of the atrioventricular node (AVN). This study was conducted, therefore, to determine the effect of acidosis on the spontaneous activity and membrane currents of myocytes isolated from the rabbit AVN, recorded at 35–37 °C using whole-cell patch-clamp. Reduction of extracellular pH (pH e; from 7.4 to 6.8 or 6.3) produced pH-dependent slowing of spontaneous action potential rate and upstroke velocity, and reductions in maximum diastolic potential and action potential amplitude. Ionic current recordings under voltage-clamp indicated that acidosis (pH e 6.3) decreased L-type Ca current ( I Ca,L), without significant changes in voltage-dependent activation or inactivation. Acidosis reduced the E-4031-sensitive, rapid delayed rectifier current ( I Kr) tail amplitude at − 40 mV following command pulses to between − 30 and + 50 mV, and accelerated tail-current deactivation. In contrast, the time-dependent hyperpolarisation-activated current, I f, was unaffected by acidosis. Background current insensitive to E-4031 and nifedipine was reduced by acidosis. Measurement of intracellular pH (pH i) from undialysed cells using BCECF showed a reduction in mean pH i from 7.24 to 6.45 ( n = 17) when pH e was lowered from 7.4 to 6.3. We conclude that I f is unlikely to be involved in the response of the AVN to acidosis, whilst inhibition of I Ca,L and I Kr by acidosis are likely to play a significant role in effects on AVN cellular electrophysiology.

Isoniazid-induced reduction in GABAergic neurotransmission alters the function of the cerebellar cortical circuit

The cerebellar cortex contributes to the control of movement, coordination, and certain cognitive functions. The cerebellar network is composed of five different types of neurons that are wired together in a repetitive module. Given that four of these five neurons synthesize and release GABA, this inhibitory neurotransmitter plays a central role in regulation of the excitability and correct functioning of the cerebellar cortex. We have now used isoniazid, an inhibitor of glutamic acid decarboxylase, the enzyme responsible for the synthesis of GABA, to evaluate the contribution of GABAergic transmission in different types of cerebellar cortical neurons to the functioning of the cerebellar circuit. Parasagittal cerebellar slices were prepared from 28- to 40-day-old male rats and were subjected to patch-clamp recording in the voltage- or current-clamp mode. Exposure of the tissue slices to isoniazid (10 mM) resulted in a decrease in the level of GABAergic transmission in Purkinje cells and a consequent increase in the firing rate of spontaneous action potentials that was apparent after 40 min. In granule neurons, isoniazid reduced both tonic and phasic GABAergic currents and thereby altered the flow of information across the cerebellar cortex. Our data support the notion that the amount of GABA at the synaptic level is a major determinant of the excitability of the cerebellar cortex, and they suggest that isoniazid may be a useful tool with which to study the function of the cerebellar network.

Role of GABA receptors in nitric oxide inhibition of dorsolateral periaqueductal gray neurons

Nitric oxide (NO) affects neuronal activity of the midbrain periaqueductal gray (PAG). The purpose of this report was to investigate the role of GABA receptors in NO modulation of neuronal activity through inhibitory and excitatory synaptic inputs within the dorsolateral PAG (dl-PAG). First, spontaneous miniature inhibitory postsynaptic currents (mIPSCs) and excitatory postsynaptic currents (mEPSCs) were recorded using whole cell voltage-clamp methods. Increased NO by either S-nitroso- N-acetyl-penicillamine (SNAP, 100 μM) or l-arginine (50 μM) significantly augmented the frequency of mIPSCs of the dl-PAG neurons without altering their amplitudes or decay time constants. The effects were eliminated after bath application of carboxy-PTIO (NO scavenger), and 1-(2-trifluorom-ethylphenyl) imidazole (NO synthase inhibitor). In contrast, SNAP and l-arginine did not alter mEPSCs in dl-PAG neurons. However the frequency of mEPSCs was significantly increased with prior application of the GABA B receptors antagonist, CGP55845. In addition, NO significantly decreased the discharge rate of spontaneous action potentials in the dl-PAG neurons and the effect was reduced in the presence of the GABA A receptor antagonist, bicuculline. Our data show that within the dl-PAG NO potentiates the synaptic release of GABA, while NO-induced GABA presynaptically inhibits glutamate release through GABA B receptors. Overall, NO suppresses neuronal activity of the dl-PAG via a potentiation of GABAergic synaptic inputs and via GABA A receptors.

BAY 41‐2272, a potent activator of soluble guanylyl cyclase, stimulates calcium elevation and calcium‐activated potassium current in pituitary GH3 cells

The effects of BAY 41-2272, a nitric oxide-independent activator of soluble guanylyl cyclase, on Ca2+ signalling and ion currents were investigated in pituitary GH3 cells. Intracellular Ca2+ concentrations ([Ca2+]i) in these cells were increased by BAY 41-2272. Removing extracellular Ca2+ abolished the BAY 41-2272-induced increase in [Ca2+]i. After [Ca2+]i was elevated by BAY 41-2272 (300 nmol/L), subsequent application of 1-benzyl-3-(5'-hydroxymethyl-2'-furyl) indazole (YC-1; 1 micromol/L) did not increase [Ca2+]i further. In whole-cell recordings, BAY 41-2272 reversibly stimulated Ca2+-activated K+ current (I(K(Ca))) with an EC50 of 225 +/- 8 nmol/L. At 3 micromol/L, BAY 41-2272 slightly and significantly decreased L-type Ca2+ current. In the cell-attached configuration, BAY 41-2272 (300 nmol/L) enhanced the activity of large-conductance Ca2+-activated K+ (BK(Ca)) channels. After BK(Ca) channel activity was stimulated by spermine NONOate (30 micromol/L) or YC-1 (10 micromol/L) in cell-attached patches, subsequent application of BAY 41-2272 (300 nmol/L) further increased the channel open probability. In the inside-out configuration, BAY 41-2272 applied to the intracellular surface of excised patches enhanced BK(Ca) channel activity. Unlike 1 micromol/L paxilline, 1H-[1,2,4]oxadiazolol-[4,3a] quinoxalin-1-one (ODQ; 10 micromol/L) or heme (10 micromol/L) had no effect on BAY 41-2272-stimulated channel activity. BAY 41-2272 caused no shift in the activation curve of BK(Ca) channels; however, it did increase the Ca2+ sensitivity of these channels. At 300 nmol/L, BAY 41-2272 reduced the firing rate of spontaneous action potentials stimulated by thyrotropin-releasing hormone (10 micromol/L). The BK(Ca) channel activity was also enhanced by 300 nmol/L BAY 41-2272 in neuroblastoma IMR-32 cells. Therefore, the BAY 41-2272-induced increase in [Ca2+]i is primarily explained by an increase in Ca2+ influx. The BAY 41-2272-mediated simulation of IK(Ca) may result from direct activation of BKCa channels and indirectly as a result of elevated [Ca2+]i.

Acute neuropharmacologic action of chloroquine on cortical neurons in vitro

Chloroquine, a common quinolone derivative used in the treatment of malaria, has been associated with neurologic side-effects including depression, psychosis and delirium. The neuropharmacologic effects of chloroquine were examined on cultured cortical neurons using microelectrode array (MEA) recording and the whole-cell patch clamp technique. Whole-cell patch clamp records under current-clamp mode also showed a chloroquine-induced depression of the firing rate of spontaneous action potentials by ∼40%, consistent with the observations with the MEA recording, although no changes in either the baseline membrane potential or input resistance were observed. Voltage clamp recordings of spontaneous post-synaptic currents, recorded in the presence of tetrodotoxin, revealed no obvious changes in either the amplitude or rate of occurrence of inward currents with application of chloroquine at 10 μM, suggesting that the fundamental molecular mechanisms underlying spontaneous synaptic transmission may not be affected by acute application of the drug. In contrast, a concentration-dependent inhibition of whole-cell calcium current was observed in the presence of chloroquine. These acute neuropharmacologic changes were not accompanied by cytotoxic actions of the compound, even after exposure of up to 500 μM chloroquine for 7 h. These data suggest that chloroquine can depress in vitro neuronal activity, perhaps through inhibition of membrane calcium channels.

Neurophysiologic Effects of Chemical Agent Hydrolysis Products on Cortical Neurons In Vitro

The neurophysiologic effects of chemical agent hydrolysis products were examined on cultured cortical neurons using multielectrode array (MEA) recording and the whole-cell patch clamp technique. Measurement of neuronal network extracellular potentials showed that the primary hydrolysis product of soman, pinacolyl methylphosphonic acid (PMPA), inhibited network mean burst and spike rates with an EC 50 of approximately 2 mM. In contrast, the degradation product of sarin, isopropyl methylphosphonic acid (IMPA), and the final common hydrolysis product of both soman and sarin, methylphosphonic acid (MPA), failed to affect neuronal network behavior at concentrations reaching 5 mM. Closer examination of the effects of PMPA (2 mM) on discriminated extracellular units revealed that mean spike amplitude was slightly diminished to 95 ± 1% (mean ± S.E.M., n = 6, P < 0.01) of control. Whole-cell patch clamp records under current clamp mode also showed a PMPA-induced depression of the firing rate of spontaneous action potentials (APs) to 36 ± 6% (n = 5, P < 0.001) of control. In addition, a minor depression with exposure to PMPA was observed in spontaneous and evoked AP amplitude to 93 ± 3% (n = 5, P < 0.05) of control with no change in either the baseline membrane potential or input resistance. Preliminary voltage clamp recordings indicated a reduction in the occurrence of spontaneous inward currents with application of PMPA. These findings suggest that PMPA, unlike MPA or IMPA, may more readily interfere with one or more aspects of excitatory synaptic transmission. Furthermore, the data demonstrate that the combination of extracellular microelectrode array and patch clamp recording techniques facilitates analysis of compounds with neuropharmacologic effects.

Vinpocetine-induced stimulation of calcium-activated potassium currents in rat pituitary GH 3 cells

The effects of vinpocetine, an inhibitor of cyclic GMP phosphodiesterase, on ionic currents were examined in rat pituitary GH 3 lactotrophs with the aid of the patch-clamp technique. In GH 3 cells bathed in normal Tyrode’s solution, vinpocetine (10 μM) reversibly increased the amplitude of Ca 2+-activated K + current ( I K(Ca)) with an ec 50 value of 4 μM. When the recording pipettes were filled with 10 mM EGTA, vinpocetine also stimulated I K(Ca). In the cell-attached configuration, application of vinpocetine to the bath increased the activity of large-conductance Ca 2+-activated K + (BK Ca) channels. In excised membrane patches, application of vinpocetine (10 μM) to the bath did not change the single-channel conductance of BK Ca channels; however, it did increase channel activity. In the inside-out configuration, neither 8-bromo cyclic GMP nor YC-1 applied intracellularly affected BK Ca channel activity. The vinpocetine-induced change in the kinetic behavior of BK Ca channels was due to an increase in mean open time and a decrease in mean closed time. Vinpocetine (10 μM) caused a leftward shift in the midpoint for the voltage-dependent opening. Under the current-clamp mode, vinpocetine (10 μM) decreased the firing rate of spontaneous action potentials induced by thyrotropin-releasing hormone (10 μM) in GH 3 cells. In pheochromocytoma PC12 cells, vinpocetine (10 μM) applied intracellularly also enhanced the activity of BK Ca channels without altering single-channel conductance. Thus, the present study suggests that vinpocetine-mediated stimulation of I K(Ca) may result from the direct activation of BK Ca channels and indirectly from elevated cytosolic Ca 2+.

Characterization of ATP-Sensitive Potassium Channels Functionally Expressed in Pituitary GH 3 Cells

ATP-sensitive K+ (KATP) channels have been characterized in pituitary GH3 cells with the aid of the patch-clamp technique. In the cell-attached configuration, the presence of diazoxide (100 microM) revealed the presence of glibenclamide-sensitive KATP channel exhibiting a unitary conductance of 74 pS. Metabolic inhibition induced by 2,4-dinitrophenol (1 mM) or sodium cyanide (300 microM) increased KATP channel activity, while nicorandil (100 microM) had no effect on it. In the inside-out configuration, Mg-ATP applied intracellularly suppressed the activity of KATP channels in a concentration-dependent manner with an IC50 value of 30 microM. The activation of phospholipase A2 caused by mellitin (1 microM) was found to enhance KATP channel activity and further application of aristolochic acid (30 microM) reduced the mellitin-induced increase in channel activity. The challenging of cells with 4,4'-dithiodipyridine (100 microM) also induced KATP channel activity. Diazoxide, mellitin and 4,4'-dithiodipyridine activated the KATP channels that exhibited similar channel-opening kinetics. In addition, under current-clamp conditions, the application of diazoxide (100 microM) hyperpolarized the membrane potential and reduced the firing rate of spontaneous action potentials. The present study clearly indicates that KATP channels similar to those seen in pancreatic beta cells are functionally expressed in GH3 cells. In addition to the presence of Ca(2+)-activated K+ channels, KATP channels found in these cells could thus play an important role in controlling hormonal release by regulating the membrane potential.