THE EFFECT OF THROMBIN IN THE SERUM-ADAPTED IONIC ENVIRONMENT ON THE INDUCTION OF EPILEPTIFORM FIRING ACTIVITY OF HIPPOCAMPAL CULTURED NEURONS

The mechanisms of epileptiform neuronal activity develop- ment under blood-brain barrier (BBB) dysfunction remains relevant in modern psychoneurology. In the present work we mimic some effects of BBB disruption in the culture of hip- pocampal neurons to examined the effect of serum-adapted ionic environment on the impulse activity of hippocampal neurons and the role of serum protein thrombin in induction of epileptiform neuronal activity. Using the whole-cell patch- clamp method under current-clamp mode we analyzed the spontaneous action potentials (AP) in the single hippocampal neurons. The changing of ionic extracellular neuronal environ- ment to such serum-adapted contributed to the development of epileptiform tonic activity of cultured hippocampal neurons and led to increase the average APs frequency by 65.1 ± 17.9% (n = 5) in neurons with spontaneous firing activity (FA) and to occurrence of tonic electrical activity (1.65 ± 0.4 s-1) in neurons without firing activity. Glutamate NMDA receptors significantly contribute to epileptiform tonic activity formation in neurons with FA, while their role in tonic activity providing in neurons without FA was insignificant. Thrombin (5 U/ml) in the serum-adapted ionic solution significantly enhanced of epileptiform activity in neurons with and without spontaneous FA: APs frequency increased in these neuronal groups by 117.3 ± 25.6% (n = 3) and by 61.8 ± 11.5% (n = 3), respective- ly, compared with that in the serum-adapted ionic solution only. Blockade of thrombin protease activated receptor 1 (PAR-1) by application of SCH 79797 (10 μm) canceled the thrombin’s effect in neurons without spontaneous FA, and significantly reduced such in neurons with FA. Therefore, the change of ionic extracellular neuronal environment to serum-adapted stimulates the occurrence of epileptiform activity in hippo- campal neurons, that is apparently associated with NMDA- receptors activation in neurons with FA. The proepileptiform action of thrombin was mostly mediated by PAR-1 activation. Thrombin-dependent regulation of the hippocampal single neurons firing activity involves the mechanisms different from the modulation of glutamate NMDA receptors in these cells.

Neurogenic substance P—influences on action potential production in afferent neurons of the kidney?

We recently showed that a substance P (SP)–dependent sympatho-inhibitory mechanism via afferent renal nerves is impaired in mesangioproliferative nephritis. Therefore, we tested the hypothesis that SP released from renal afferents inhibits the action potential (AP) production in their dorsal root ganglion (DRG) neurons. Cultured DRG neurons (Th11-L2) were investigated in current clamp mode to assess AP generation during both TRPV1 stimulation by protons (pH 6) and current injections with and without exposure to SP (0.5 µmol) or CGRP (0.5 µmol). Neurons were classified as tonic (sustained AP generation) or phasic (≤ 4 APs) upon current injection; voltage clamp experiments were performed for the investigation of TRPV1-mediated inward currents due to proton stimulation. Superfusion of renal neurons with protons and SP increased the number of action potentials in tonic neurons (9.6 ± 5 APs/10 s vs. 16.9 ± 6.1 APs/10 s, P < 0.05, mean ± SD, n = 7), while current injections with SP decreased it (15.2 ± 6 APs/600 ms vs. 10.2 ± 8 APs/600 ms, P < 0.05, mean ± SD, n = 29). Addition of SP significantly reduced acid-induced TRPV1-mediated currents in renal tonic neurons (− 518 ± 743 pA due to pH 6 superfusion vs. − 82 ± 50 pA due to pH 6 with SP superfusion). In conclusion, SP increased action potential production via a TRPV1-dependent mechanism in acid-sensitive renal neurons. On the other hand, current injection in the presence of SP led to decreased action potential production. Thus, the peptide SP modulates signaling pathways in renal neurons in an unexpected manner leading to both stimulation and inhibition of renal neuronal activity in different (e.g., acidic) environmental contexts.

Hybrid quantum dot - collagen extracellular matrices for in situ optical monitoring of cardiomyocyte activity by two-photon fluorescence

ABSTRACTThe incorporation of functional nanoparticles in scaffolds for tissue constructs has led to the creation of artificial extracellular matrices that more accurately mimic the cues present in the native microenvironment of developing tissue. Additionally, light-sensitive inorganic nanoparticles can act as cell biosensors and report on the physiological parameters during tissue growth and organization. In this work, we functionalized collagen nanofibers with semiconductor quantum dots (QDs) and thereby created artificial extracellular matrices that can optically report on cardiomyocyte activity based on QD two-photon fluorescence. We have applied these optically-addressable nanofiber matrices to monitor activities of primary cardiomyocytes and compared the optical responses with patch-clamp data. Combining the long-term stability of QD fluorescence with the deeper light penetration depths achievable through multiphoton imaging, this approach can be used for continuous monitoring of cellular functions in cardiac tissue engineering.Abstract FigureConcept illustration: optical readout of cardiomyocyte activity with QD-functionalized collagen networks. Whole-cell current-clamp mode is used here to simultaneously monitor changes in the transmembrane voltage while the QD two-photon fluorescence is recorded.

Decreased excitability and voltage-gated sodium currents in aortic baroreceptor neurons contribute to the impairment of arterial baroreflex in cirrhotic rats

Cardiovascular autonomic dysfunction, which is manifested by an impairment of the arterial baroreflex, is prevalent irrespective of etiology and contributes to the increased morbidity and mortality in cirrhotic patients. However, the cellular mechanisms that underlie the cirrhosis-impaired arterial baroreflex remain unknown. In the present study, we examined whether the cirrhosis-impaired arterial baroreflex is attributable to the dysfunction of aortic baroreceptor (AB) neurons. Biliary and nonbiliary cirrhotic rats were generated via common bile duct ligation (CBDL) and intraperitoneal injections of thioacetamide (TAA), respectively. Histological and molecular biological examinations confirmed the development of fibrosis in the livers of both cirrhotic rat models. The heart rate changes during phenylephrine-induced baroreceptor activation indicated that baroreflex sensitivity was blunted in the CBDL and TAA rats. Under the current-clamp mode of the patch-clamp technique, cell excitability was recorded in DiI-labeled AB neurons. The number of action potential discharges in the A- and C-type AB neurons was significantly decreased because of the increased rheobase and threshold potential in the CBDL and TAA rats compared with sham-operated rats. Real-time PCR and Western blotting indicated that the NaV1.7, NaV1.8, and NaV1.9 transcripts and proteins were significantly downregulated in the nodose ganglion neurons from the CBDL and TAA rats compared with the sham-operated rats. Consistent with these molecular data, the tetrodotoxin-sensitive NaV currents and the tetrodotoxin-resistant NaV currents were significantly decreased in A- and C-type AB neurons, respectively, from the CBDL and TAA rats compared with the sham-operated rats. Taken together, these findings implicate a key cellular mechanism in the cirrhosis-impaired arterial baroreflex.

Frequency-selective transmission of graded signals in large monopolar neurons of blowfly Calliphora vicina compound eye

The functional roles of voltage-gated K+ (Kv) channels in visual system interneurons remain poorly studied. We have addressed this problem in the large monopolar cells (LMCs) of the blowfly Calliphora vicina, using intracellular recordings and mathematical modeling methods. Intracellular recordings were performed in two cellular compartments: the synaptic zone, which receives input from photoreceptors, and the axon, which provides graded potential output to the third-order visual neurons. Biophysical properties of Kv conductances in the physiological voltage range were examined in the dark with injections of current in the discontinuous current-clamp mode. Putative LMC types 1/2 and 3 (L1/2 and L3, respectively) had dissimilar Kv channelomes: L1/2 displayed a prominent inactivating Kv conductance in the axon, while L3 cells were characterized by a sustained delayed-rectifier Kv conductance. To study the propagation of voltage signals, the data were incorporated into the previously developed mathematical model. We demonstrate that the complex interaction between the passive membrane properties, Kv conductances, and the neuronal geometry leads to a resonance-like filtering of signals with peak frequencies of transmission near 15 and 40 Hz for L3 and L1/2, respectively. These results point to distinct physiological roles of different types of LMCs.

Effect of a novel BKCa opener on BKCa currents and contractility of the rabbit corpus cavernosum

Large-conductance Ca2+-activated K+ (BKCa) channels are thought to play a key role in the regulation of corpus cavernosum smooth muscle (CCSM) excitability. Few BKCa channel openers have been accepted for clinical development. The effect of the novel BKCa channel opener GoSlo-SR5-130 on electrical activity in isolated rabbit CCSM cells and mechanical activity in strips of rabbit CCSM was examined. Single-channel currents were observed in inside-out patches. These channels were sensitive to Ca2+, blocked by penitrem A, and had a conductance of 291 ± 20 pS ( n = 7). In the presence of GoSlo-SR5-130, the number of open BKCa channels increased. Using voltage-ramp protocols, GoSlo-SR5-130 caused currents to activate at more negative potentials in a concentration-dependent manner, shifting the half-maximal activation voltage potential to the left on the voltage axis. Therefore, BKCa channels were open within the physiological range of membrane potentials in the presence of GoSlo-SR5-130. GoSlo-SR5-130 also resulted in an increase in the activity of spontaneous transient outward currents in myocytes isolated from CCSM, and this effect was reversed by iberiotoxin. In current-clamp mode, GoSlo-SR5-130 hyperpolarized the cell membrane. Isometric tension recording of strips of rabbit corpus cavernosum showed that GoSlo-SR5-130 inhibited spontaneous contractions in a concentration-dependent manner. This effect was reversed in the presence of iberiotoxin, suggesting that GoSlo-SR5-130 exerts its effect through BKCa channels. These findings suggest that GoSlo-SR5-130 is an effective tool for the study of BKCa channels and that these channels can modulate CCSM activity and are possible targets for the treatment of erectile dysfunction.

Evidence for the Inhibition by Temozolomide, an Imidazotetrazine Family Alkylator, of Intermediate-Conductance Ca2+-Activated K+ Channels in Glioma Cells

Background: Temozolomide (TMZ), an oral alkylator of the imidazotetrazine family, is used to treat glioma. Whether this drug has any ionic effects in glioma cells remains largely unclear. Methods: With the aid of patch-clamp technology, we investigated the effects of TMZ on the ionic currents in U373 glioma cells. The mRNA expression of KCNN4 (KCa3.1) in U373 glioma cells and TMZ's effect on K+ currents in these KCNN4 siRNA-transfected U373 cells were investigated. Results: In whole-cell recordings, TMZ decreased the amplitude of voltage-dependent K+ currents (IK) in U373 cells. TMZ-induced IK inhibition was reversed by ionomycin or 1-ethyl-2-benzimidazolinone (1-EBIO). In cell-attached configuration, TMZ concentration-dependently reduced the activity of intermediate-conductance Ca2+-activated K+ (IKCa) channels with an IC50 value of 9.2 µM. Chlorzoxazone or 1-EBIO counteracted the TMZ-induced inhibition of IKCa channels. Although TMZ was unable to modify single-channel conductance, its inhibition of IKCa channels was weakly voltage-dependent and accompanied by a significant prolongation in the slow component of mean closed time. However, neitherlarge-conductance Ca2+-activated (BKCa) nor inwardly rectifying K+ (Kir) channels were affected by TMZ. In current-clamp mode, TMZ depolarized the cell membrane and 1-EBIO reversed TMZ-induced depolarization. TMZ had no effect on IK in KCNN4 siRNA-transfected U373 cells. Conclusion: In addition to the DNA damage it does, its inhibitory effect on IKCa channels accompanied by membrane depolarization could be an important mechanism underlying TMZ-induced antineoplastic actions.

Mechanotransduction and hyperpolarization-activated currents contribute to spontaneous activity in mouse vestibular ganglion neurons

The hyperpolarization-activated, cyclic nucleotide–sensitive current, Ih, is present in vestibular hair cells and vestibular ganglion neurons, and is required for normal balance function. We sought to identify the molecular correlates and functional relevance of Ih in vestibular ganglion neurons. Ih is carried by channels consisting of homo- or heteromeric assemblies of four protein subunits from the Hcn gene family. The relative expression of Hcn1–4 mRNA was examined using a quantitative reverse transcription PCR (RT-PCR) screen. Hcn2 was the most highly expressed subunit in vestibular neuron cell bodies. Immunolocalization of HCN2 revealed robust expression in cell bodies of all vestibular ganglion neurons. To characterize Ih in vestibular neuron cell bodies and at hair cell–afferent synapses, we developed an intact, ex vivo preparation. We found robust physiological expression of Ih in 89% of cell bodies and 100% of calyx terminals. Ih was significantly larger in calyx terminals than in cell bodies; however, other biophysical characteristics were similar. Ih was absent in calyces lacking Hcn1 and Hcn2, but small Ih was still present in cell bodies, which suggests expression of an additional subunit, perhaps Hcn4. To determine the contributions of hair cell mechanotransduction and Ih to the firing patterns of calyx terminals, we recorded action potentials in current-clamp mode. Mechanotransduction currents were modulated by hair bundle defection and application of calcium chelators to disrupt tip links. Ih activity was modulated using ZD7288 and cAMP. We found that both hair cell transduction and Ih contribute to the rate and regularity of spontaneous action potentials in the vestibular afferent neurons. We propose that modulation of Ih in vestibular ganglion neurons may provide a mechanism for modulation of spontaneous activity in the vestibular periphery.

Involvement of MAPKs and PLC Pathways in Modulation of Pacemaking Activity by So-Cheong-Ryong-Tang in Interstitial Cells of Cajal from Murine Small Intestine

Purpose. Interstitial cells of Cajal (ICCs) are the pacemaker cells that generate slow waves in the gastrointestinal (GI) tract. We have aimed to investigate the effects of Socheongryong-Tang (SCRT) in ICCs from mouse’s small intestine.Methods. The whole-cell patch-clamp configuration was used to record membrane potentials from cultured ICCs. Intracellular Ca2+([Ca2+]i) increase was studied in cultured ICCs using fura-2 AM.Results. ICCs generated pacemaker potentials in mouse’s small intestine. SCRT produced membrane depolarization in current clamp mode. Y25130 (5-HT3receptor antagonist) and RS39604 (5-HT4receptor antagonist) blocked SCRT-induced membrane depolarizations, whereas SB269970 (5-HT7receptor antagonist) did not. When GDP-β-S (1 mM) was in the pipette solution, SCRT did not induce the membrane depolarizations.[Ca2+]ianalysis showed that SCRT increased[Ca2+]i. In the presence of PD98059 (p42/44 MAPK inhibitor), SCRT did not produce membrane depolarizations. In addition, SB203580 (p38 MAPK inhibitor) and JNK inhibitors blocked the depolarizations by SCRT in pacemaker potentials. Furthermore, the membrane depolarizations by SCRT were not inhibited by U-73122, an active phospholipase C (PLC) inhibitor, but by U-73343, an inactive PLC inhibitor.Conclusion. These results suggest that SCRT might affect GI motility by the modulation of pacemaker activity through MAPKs and PLC pathways in the ICCs.

Suppression of human detrusor smooth muscle excitability and contractility via pharmacological activation of large conductance Ca2+-activated K+channels

Overactive bladder syndrome is frequently associated with increased detrusor smooth muscle (DSM) contractility. We tested the hypothesis that pharmacological activation of the large-conductance voltage- and Ca2+-activated K+(BK) channel with NS-1619, a selective BK channel opener, reduces the excitability and contractility of human DSM. We used the amphotericin-perforated whole cell patch-clamp technique on freshly isolated human DSM cells, live-cell Ca2+imaging, and isometric DSM tension recordings of human DSM strips obtained from open bladder surgeries. NS-1619 (30 μM) significantly increased the amplitude of the voltage step-induced whole cell BK currents, and this effect was abolished by pretreatment with 200 nM iberiotoxin (IBTX), a selective BK channel inhibitor. In current-clamp mode, NS-1619 (30 μM) significantly hyperpolarized the resting membrane potential, and the hyperpolarization was reversed by IBTX (200 nM). NS-1619 (30 μM) significantly decreased the intracellular Ca2+level in isolated human DSM cells. BK channel activation with NS-1619 (30 μM) significantly inhibited the amplitude, muscle force, frequency, duration, and tone of the spontaneous phasic and pharmacologically induced DSM contractions from human DSM isolated strips. IBTX (200 nM) suppressed the inhibitory effects of NS-1619 on spontaneous contractions. The amplitude of electrical field stimulation (0.5–50 Hz)-induced contractions was significantly reduced by NS-1619 (30 μM). Our data suggest that pharmacological activation of BK channels could represent a novel treatment option to control bladder dysfunction in humans.