Melatonin inhibits arrhythmias induced by increased late sodium currents in ventricular myocytes / 药学学报

Melatonin (Mel) has been shown to have cardioprotective effects, but its action on ion channels is unclear. In this experiment, we investigated the inhibitory effect of Mel on late sodium currents (INa.L) in mouse ventricular myocytes and the anti-arrhythmic effect at the organ level as well as its mechanism. The whole-cell patch clamp technique was applied to record the ionic currents and action potential (AP) in mouse ventricular myocytes while the electrocardiogram (ECG) and monophasic action potential (MAP) were recorded simultaneously in mouse hearts using a multichannel acquisition and analysis system. The results demonstrated that the half maximal inhibitory concentration (IC50) values of Mel on transient sodium current (INa.T) and specific INa.L opener 2 nmol·L-1 sea anemone toxins II (ATX II) increased INa.L were 686.615 and 7.37 μmol·L-1, respectively. Mel did not affect L-type calcium current (ICa.L), transient outward current (Ito), and AP. In addition, 16 μmol·L-1 Mel shortened ATX II-prolonged action potential duration (APD), suppressed ATX II-induced early afterdepolarizations (EADs), and significantly reduced the incidence of ventricular tachycardia (VT) and ventricular fibrillation (VF) in Langendorff-perfused mouse hearts. In conclusion, Mel exerted its antiarrhythmic effects principally by blocking INa.L, thus providing a significant theoretical basis for new clinical applications of Mel. Animal welfare and experimental process are in accordance with the regulations of the Experimental Animal Ethics Committee of Wuhan University of Science and Technology (2023130).

Effects of propofol on glutamatergic neuronal activity in paraventricular thalamus / 药学学报

This study explored the effects of propofol on the activity of glutamatergic neurons in the paraventricular thalamus (PVT) and the underlying mechanisms at the molecular level using whole-cell patch-clamp techniques. Acute brain slices containing the PVT were obtained from 8 weeks old C57BL/6J mice. The electrophysiological characteristics of PVT neurons were recorded in current-clamp mode, then single-cell sequencing was used to identify neuronal types. The firing frequencies before, during, and after propofol or intralipid application were recorded as FB, FD and FW; and the membrane potentials were recorded as MPB and MPD. Picrotoxin (PTX) was used to block inhibitory gamma-aminobutyric acid type A (GABAA) receptors during the application of propofol at 10 μmol·L-1. Then, GABAA receptor-mediated spontaneous and miniature inhibitory postsynaptic currents (sIPSCs and mIPSCs) were recorded, and the effects of 10 μmol·L-1 propofol were investigated. The animal experiments were approved by the Medical Animal Administrative Committee of Shanghai Medical College Fudan University. The results showed that there were no significant differences in FB, FD and FW during intralipid and 2 μmol·L-1 propofol application. With propofol at 5, 10 and 20 μmol·L-1, FD decreased significantly when compared with FB, and FW increased significantly as compared with FD (P < 0.01). The inhibition degree of the three concentration groups was significantly different (P < 0.01). In addition, with propofol at 20 μmol·L-1, MPD hyperpolarized significantly (P < 0.01). In the presence of PTX, 10 μmol·L-1 propofol could not suppress the firing frequency of PVT glutamatergic neurons. Propofol at 10 μmol·L-1 prolonged the decay time of sIPSCs (P < 0.01) and mIPSCs (P < 0.05), and increased the amplitude (P < 0.01) of mIPSCs of PVT glutamatergic neurons. Together, these results indicate that propofol can inhibit the activity of PVT glutamatergic neurons in a concentration-dependent and reversible manner, and the effect is likely to be mediated by postsynaptic GABAA receptors.

Ferulic acid enhances insulin secretion by potentiating L-type Ca2+ channel activation / 中西医结合学报

OBJECTIVE@#To investigate the effect of ferulic acid, a natural compound, on pancreatic beta cell viability, Ca2+ channels, and insulin secretion.@*METHODS@#We studied the effects of ferulic acid on rat insulinoma cell line viability using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide viability assay. The whole-cell patch-clamp technique and enzyme-linked immunosorbent assay were also used to examine the action of ferulic acid on Ca2+ channels and insulin secretion, respectively.@*RESULTS@#Ferulic acid did not affect cell viability during exposures up to 72 h. The electrophysiological study demonstrated that ferulic acid rapidly and concentration-dependently increased L-type Ca2+ channel current, shifting its activation curve in the hyperpolarizing direction with a decreased slope factor, while the voltage dependence of inactivation was not affected. On the other hand, ferulic acid have no effect on T-type Ca2+ channels. Furthermore, ferulic acid significantly increased insulin secretion, an effect inhibited by nifedipine and Ca2+-free extracellular fluid, confirming that ferulic acid-induced insulin secretion in these cells was mediated by augmenting Ca2+ influx through L-type Ca2+ channel. Our data also suggest that this may be a direct, nongenomic action.@*CONCLUSION@#This is the first electrophysiological demonstration that acute ferulic acid treatment could increase L-type Ca2+ channel current in pancreatic β cells by enhancing its voltage dependence of activation, leading to insulin secretion.

Suxiao Jiuxin Pills Prevent Ventricular Fibrillation from Inhibiting L-type Calcium Currents CaV1.2 in vivo and in vitro / 中国结合医学杂志

OBJECTIVE@#To investigate whether Suxiao Jiuxin Pills (SJP), a Chinese herbal remedy, is an anti-ventricular fibrillation (VF) agent.@*METHODS@#VF was induced by isoproterenolol (ISO) intraperitoneal injection followed by electrical pacing in mice and rabbits. The effects of SJP on the L-type calcium channel current (CaV1.2), voltage-dependent sodium channel current (INa), rapid and slow delayed rectifier potassium channel current (IKr and IKs, respectively) were studied by whole-cell patch-clamp method. Computer simulation was implemented to incorporate the experimental data of SJP effects on the CaV1.2 current into the action potential (AP) and pseudo-electrocardiography (pseudo-ECG) models.@*RESULTS@#SJP prevented VF induction and reduced VF durations significantly in mice and rabbits. Patch-clamp experiments revealed that SJP decreased the peak amplitude of the CaV1.2 current with a half maximal concentration (IC50) value of 16.9 mg/L (SJP-30 mg/L, -32.8 ± 6.1 pA; Verapamil, -16.2 ±1.8 pA; vs. control, -234.5 ±16.7 pA, P<0.01, respectively). The steady-state activation curve, inactivation curve, and the recovery from inactivation of the CaV1.2 current were not shifted significantly. Specifically, SJP did not altered INa, IKr, and IKs currents significantly (SJP vs. control, P>0.05). Computer simulation showed that SJP-reduced CaV1.2 current shortened the AP duration, transiting VF into sinus rhythm in pseudo-ECG.@*CONCLUSION@#SJP reduced VF via inhibiting the CaV1.2 current with in vivo, in vitro, and in silico studies, which provide experimental basis for SJP anti-VF clinical application.

Effects of early sleep deprivation on depressive-like behavior and hippocampus synaptic plasticity in adult depression model mice / 中华行为医学与脑科学杂志

Objective:To investigate the effects of early sleep deprivation(SD) on depressive-like behavior and hippocampus synaptic plasticity in adult mice with chronic unpredictable mild stress(CUMS) model.Methods:Thirty 2-week-old clean grade male mice were randomly divided into control group (CON group), CUMS group and SD + CUMS group according to the random number table, with 10 mice in each group. The mice in SD + CUMS group were subjected with sleep deprivation for 4 hours once a day during puberty (3 ~ 6 weeks old), and then were stimulated by CUMS after adulthood (9 weeks old). The mice in CUMS group were subjected with CUMS at the age of 9 weeks. And the mice in CON group were not given any intervention.The depressive-like behavior was evaluated by body weight, sugar water preference, tail suspension test and forced swimming test.The density of dendritic spines of basal and apical neurons in hippocampal CA1 was measured by Golgi staining, the frequency and amplitude of miniature excitatory postsynaptic current(mEPSC) of pyramidal neurons in the hippocampal CA1 region of mice were measured by electro-physiological patch clamp technique.Graphpad prism 7.0 software was used for statistical analysis and mapping. One-way ANOVA was used for comparison among multiple groups, and Tukey test was used for further pairwise comparison.Results:(1) After stress modeling, there were significant differences in body weight, sugar water preference percentage, forced swimming immobility time and tail suspension time among the three groups ( F=71.63, 39.82, 44.13, 43.07, all P<0.01). Compared with CON group, the mice in CUMS group and SD+ CUMS group had lower body weight ((25.51±0.37) g, (22.92±0.31) g, (20.12±0.27) g, both P<0.01), lower sugar water percentage preference ((87.40±1.65) %, (63.42±3.33) %, (49.68±3.70)%, both P<0.01), longer immobile time of forced swimming ((34.30±5.32) s, (119.20±12.03) s, (153.80±9.17) s, both P<0.01) and longer immobile time of tail suspension test((115.20±8.19)s, (156.80±4.35) s, (192.00±4.12) s, both P<0.01). Compared with CUMS group, SD+ CUMS group had lower body weight ( P<0.01), lower sugar water preference percentage ( P<0.05), longer immobile time in forced swimming test( P<0.05) and longer immobile time in tail suspension test( P<0.01). (2) Golgi staining results showed that the densities of dendritic spines of apical neurons and basal neurons in hippocampal CA1 area of the three groups were significantly different ( F=38.41, 41.34, both P<0.01). The densities of dendritic spines of basal and apical hippocampal neurons in CUMS group and SD+ CUMS group were lower than those in CON group ((7.74±0.22)/10 μm, (6.58±0.27)/10 μm, (5.00±0.13)/10 μm, both P<0.01), ((8.90±0.23)/10 μm, (7.63±0.30)/10 μm, (6.01±0.14)/10 μm, both P<0.01). Compared with CUMS group, the mice in SD+ CUMS group had lower densities of dendritic spines of basal and apical hippocampal neurons(both P<0.01). (3) Electrophysiological results showed that there were significant differences in the frequency and amplitude of mEPSC in hippocampal pyramidal neurons of the three groups ( F=38.90, 63.37, both P<0.01). Compared with CON group, the frequency and amplitude of mEPSC in pyramidal neurons of CA1 in CUMS group and SD+ CUMS group were significantly lower ((0.39±0.03)Hz, (0.20±0.02)Hz, (0.07±0.02)Hz, both P<0.01; (9.98±0.31)pA, (7.74±0.21)pA, 6.36±0.13)pA, both P<0.01). Compared with CUMS group, the frequency and amplitude of mEPSC in SD+ CUMS group were lower (both P<0.01). Conclusion:Adolescent sleep deprivation aggravates depressive behavior and hippocampus synaptic plasticity impairment in adult CUMS model mice.

Effect of Sodium Salicylate on Calcium Currents and Exocytosis in Cochlear Inner Hair Cells: Implications for Tinnitus Generation / 神经科学通报·英文版

Sodium salicylate is an anti-inflammatory medication with a side-effect of tinnitus. Here, we used mouse cochlear cultures to explore the effects of salicylate treatment on cochlear inner hair cells (IHCs). We found that IHCs showed significant damage after exposure to a high concentration of salicylate. Whole-cell patch clamp recordings showed that 1-5 mmol/L salicylate did not affect the exocytosis of IHCs, indicating that IHCs are not involved in tinnitus generation by enhancing their neuronal input. Instead, salicylate induced a larger peak amplitude, a more negative half-activation voltage, and a steeper slope factor of Ca2+ current. Using noise analysis of Ca2+ tail currents and qRT-PCR, we further found that salicylate increased the number of Ca2+ channels along with CaV1.3 expression. All these changes could act synergistically to enhance the Ca2+ influx into IHCs. Inhibition of intracellular Ca2+ overload significantly attenuated IHC death after 10 mmol/L salicylate treatment. These results implicate a cellular mechanism for tinnitus generation in the peripheral auditory system.

Isolation, identification and electrophysiological activity of BmK M2 from Buthus martensii Karsch venom / 中国药科大学学报

@#This study aimed to isolate and identify novel toxin peptides targeting voltage-gated sodium channels (VGSGs) from the venom of the Buthus martensii Karsch (BmK) scorpion. Using G50-gel filtration, HPLC, peptide fingerprinting and amino acid sequencing, a novel sodium channel modulator, BmK M2, was identified from BMK scorpion. BmK M2 is a relatively abundant long chain polypeptide toxin in BmK scorpion venom with a molecular weight of 7 235.59, consisting of 64 amino acids and 4 pairs of disulfide bonds.Sequence alignment showed that the amino acid sequence of BmK M2 had high sequence and structural similarity to that of the discovered sodium channel toxins of BmK M1, BmK M3 and BmK M9, etc.BmK M2 is a potential new sodium channel modulator.Electrophysiological results revealed that BmK M2 can significantly enhance the activation, delay the steady-state inactivation and closed-state inactivation of Nav1.7, but has no activity on Nav1.8.BmK M2 can be used as a novel peptide probe for the study of the structure and function of Nav1.7 and the development of drugs targeting Nav1.7.

Study on the temperature characteristics of fast capacitance in patch clamp experiments / 生物医学工程学杂志

Patch clamp is a technique that can measure weak current in the level of picoampere (pA). It has been widely used for cellular electrophysiological recording in fundamental medical researches, such as membrane potential and ion channel currents recording, etc. In order to obtain accurate measurement results, both the resistance and capacitance of the pipette are required to be compensated. Capacitance compensations are composed of slow and fast capacitance compensation. The slow compensation is determined by the lipid bilayer of cell membrane, and its magnitude usually ranges from a few picofarads (pF) to a few microfarads (μF), depending on the cell size. The fast capacitance is formed by the distributed capacitance of the glass pipette, wires and solution, mostly ranging in a few picofarads. After the pipette sucks the cells in the solution, the positions of the glass pipette and wire have been determined, and only taking once compensation for slow and fast capacitance will meet the recording requirements. However, when the study needs to deal with the temperature characteristics, it is still necessary to make a recognition on the temperature characteristic of the capacitance. We found that the time constant of fast capacitance discharge changed with increasing temperature of bath solution when we studied the photothermal effect on cell membrane by patch clamp. Based on this phenomenon, we proposed an equivalent circuit to calculate the temperature-dependent parameters. Experimental results showed that the fast capacitance increased in a positive rate of 0.04 pF/℃, while the pipette resistance decreased. The fine data analysis demonstrated that the temperature rises of bath solution determined the kinetics of the fast capacitance mainly by changing the inner solution resistance of the glass pipette. This result will provide a good reference for the fine temperature characteristic study related to cellular electrophysiology based on patch clamp technique.

Effect of etomidate on the reuniens neuronal activity / 中南大学学报(医学版)

OBJECTIVES@#To explore the effect of etomidate on the neuronal activity of ventral thalamic reuniens nucleus and the underlying mechanisms.@*METHODS@#Whole-cell patch clamp method was used to explore the effect of etomidate on the activity of ventral thalamic reuniens neurons in the acute brain slices obtained from 4-5 weeks old C57BL/6J mice. The electrophysiological characteristics of ventral thalamic reuniens neurons were recorded in the current clamp mode, and then the effects of etomidate (0.5, 2.0, 8.0 μmol/L etomidate groups) and intralipid (intralipid group) on the discharge frequency and membrane potential of ventral thalamic reuniens neurons were recorded. During the experiment, the ventral thalamic reuniens neuron firing rates (RNFRs) were recorded as F@*RESULTS@#In the intralipid group, there was no significant difference among the F@*CONCLUSIONS@#Etomidate can inhibit the activity of ventral thalamic reuniens neurons in concentration-dependent manner, and which is reversible. Etomidate with sub-anesthetic concentration inhibits the activity of ventral thalamic reuniens neurons via targeting the GABA

Jingzhaotoxin-X, a gating modifier of Kv4.2 and Kv4. 3 potassium channels purified from the venom of the Chinese tarantula Chilobrachys jingzhao

The tarantula Chilobrachys jingzhao is one of the largest venomous spiders in China. In previous studies, we purified and characterized at least eight peptides from C. jingzhao venom. In this report, we describe the purification and characterization of Jingzhaotoxin-X (JZTX-X), which selectively blocks Kv4.2 and Kv4.3 potassium channels. Methods: JZTX-X was purified using a combination of cation-exchange HPLC and reverse-phase HPLC. The amino-acid sequence was determined by automated Edman degradation and confirmed by mass spectrometry (MS). Voltage-gated ion channel currents were recorded in HEK293t cells transiently transfected with a variety of ion channel constructs. In addition, the hyperalgesic activity of JZTX-X and the toxin´s effect on motor function were assessed in mice. Results: JZTX-X contained 31 amino acids, with six cysteine residues that formed three disulfide bonds within an inhibitory cysteine knot (ICK) topology. In whole-cell voltage-clamp experiments, JZTX-X inhibited Kv4.2 and Kv4.3 potassium channels in a concentration- and voltage-dependent manner, without affecting other ion channels (Kv1.1, 1.2, 1.3, 2.1, delayed rectifier potassium channels, high- and low-voltage-activated Ca2+ channels, and voltage-gated sodium channels Nav1.5 and 1.7). JZTX-X also shifted the voltage-dependent channel activation to more depolarized potentials, whereas extreme depolarization caused reversible toxin binding to Kv4.2 channels. JZTX-X shifted the Kv4.2 and Kv4.3 activities towards a resting state, since at the resting potential the toxin completely inhibited the channels, even in the absence of an applied physical stimulus. Intrathecal or intraplantar injection of JZTX-X caused a long-lasting decrease in the mechanical nociceptive threshold (hyperalgesia) but had no effect on motor function as assessed in the rotarod test. Conclusions: JZTX-X selectively suppresses Kv4.2 and Kv4.3 potassium channel activity in a concentration- and voltage-dependent manner and causes long-lasting mechanical hyperalgesia.(AU)

Effects of bone morphogenetic protein 2 and basic fibroblast growth factor 2 on proliferation and osteogenic differentiation of rat bone marrow mesenchymal stem cell sheet / 中国组织工程研究

BACKGROUND: The effects and mechanisms of bone morphogenetic protein 2 and basic fibroblast growth factor 2 on the proliferation and osteogenic differentiation of bone mesenchymal stem cells still remain unknown. How to combine the growth factors with tissue-engineered cell patch clamp techniques is of great significance for bone defect repair. OBJECTIVE: To explore the effects of bone morphogenetic protein 2 and basic fibroblast growth factor 2 applied alone or in combination on the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cell sheet. METHODS: Bone marrow mesenchymal stem cells from Sprague-Dawley rats were isolated, cultured and identified in vitro to construct cell sheet. Bone morphogenetic protein 2 and basic fibroblast growth factor 2 at different concentrations were individually or jointly used to induce bone marrow mesenchymal stem cell sheet. The cell counting kit-8 assay combined with alkaline phosphatase activity assay was used to determine the optimal concentration of the two factors in promoting cell proliferation and osteogenic differentiation. Osteogenic induction of bone marrow mesenchymal stem cell sheet was assessed by gross and microscopic observations, Vonkossa staining, alizarin red staining, and RT-PCR detection. RESULTS AND CONCLUSION: The single application of bone morphogenetic protein 2 enhanced the alkaline phosphatase activity of the bone marrow mesenchymal stem cell sheet, and the optimal concentration was 100 μg/L (P < 0.001). The single application of basic fibroblast growth factor 2 accelerated the proliferation of bone marrow mesenchymal stem cell sheet, and the optimal concentration was 20 μg/L (P < 0.001). Their combination facilitated the proliferation of the cell sheet, and boosted the alkaline phosphatase activities (P < 0.001). The four groups of cell sheet showed no significant morphological difference, and the osteogenic differentiation of the bone marrow mesenchymal stem cell sheet could all be induced through the osteogenic induction. Calcium nodules were most significant in the combination group (P < 0.001), suggesting that the combination significantly facilitated late osteogenic differentiation, suppressed early osteogenic differentiation of the sheet and showed significant synergistic effect (P < 0.001). In summary, the application of bone morphogenetic protein 2 combined with basic fibroblast growth factor 2 plays a synergistic role in promoting the proliferation of bone marrow mesenchymal stem cell sheet and significantly enhances the osteogenic induction.

Diethylmorphine effect on action potential and calcium ion current of neonatal rat cardiomyocytes in vitro / 中国组织工程研究

BACKGROUND: Opioids can regulate the changes of membrane potential and Ca2+ current in cardiomyocytes, but whether diacetylmorphine can induce the changes of cardiac rhythm, cell action potential and Ca2+ current has not been reported. OBJECTIVE: To explore the effect of diacetylmorphine on action potential and calcium current of isolated cardiomyocytes from neonatal Sprague-Dawley rats. METHODS: Five concentrations of diacetylmorphine (0, 10-2, 10-3, 10-4, 10-5 mol/L) and 20 mol/L verapamil were used to treat the cardiomyocytes of neonatal Sprague-Dawley rats cultured in vitro. The cells were divided into control group, diacetylmorphine group, diacetylmorphine+verapamil group. The latter two groups were treated with diacetylmorphine and diacetylmorphine+verapamil (20 μmol/L), respectively, while the control group was treated with the same amount of PBS. The study protocol was approved by the Animal Ethics Committee of the First Affiliated Hospital of Xinjiang Medical University on May 21, 2018 with approval No. IACUC201805-K1. RESULTS AND CONCLUSION: At 24 hours of culture with different concentrations of diacetylmorphine, the number of cardiomyocytes with abnormal morphology increased significantly in a dose-dependent manner. When the concentration of diacetylmorphine increased, the number of survived cells decreased, with a reduction in the size of cytoplasm and number of pseudopods, the cell membrane was shrunk and the nuclear structure was blurred. Compared with the control group, when diacetylmorphine was added to intervene with the cardiomyocytes, there was a significant difference in the spontaneous beating frequency and rhythm of cardiomyocytes. The negative value of resting membrane potential decreased, while the time course of action potential increased significantly, and the amplitude of action potential decreased significantly. Compared with the control group, the number of cells with changes in the membrane potential significantly increased in the diacetylmorphine group. The addition of verapamil reduced the number of cells with changes in the membrane potential. Compared with the control group, the number of cells with variation of membrane potential was increased to some extents. These findings suggest that diacetylmorphine can induce cardiomyocyte morphological abnormality, increase the spontaneous beating frequency and rhythm of cardiomyocytes, and change the membrane potential and action potential of cardiomyocytes. Verapamil acts as a calcium channel blocker that can improve the rhythm abnormality of cardiomyocytes induced by diacetylmorphine.

Characteristics of electrophysiological changes in the process of astrocytes pyroptosis after hyperoxia exposure / 中南大学学报(医学版)

OBJECTIVES@#To observe the electrophysiological changes of astrocytes in the process of hyperoxia induced apoptosis and analyze the relationship between electrophysiological characteristics and morphological changes.@*METHODS@#Astrocytes were exposed to 90% hyperoxia for 12-72 h. The electrophysiological characteristics of astrocytes in each group were detected by patch clamp technique, and the morphological characteristics of astrocytes were observed at the same time. Then the same batch of astrocytes were collected, and the expression levels of caspase-1, caspase-3, gasdermin D (GSDMD) and gasdermin E (GSDME) were detected by Western blotting.@*RESULTS@#From 12 h to 72 h after hyperoxia exposure, the inward current was significantly lower than that of the control group (0.05). At each time point, the morphology of cells changed correspondingly. Western blotting showed that the expression of caspase-1 was increased significantly at 24 h and decreased significantly at 72 h after hyperoxia exposure (0.05), but began to decrease at 48 h (<0.05); GSDME increased gradually at 24 h after hyperoxia exposure (<0.05).@*CONCLUSIONS@#Under hyperoxia exposure, the ion channels of astrocytes are damaged, which can maintain the dysfunction of ion homeostasis, activate GSDME, induce the damaged cells to break away from the apoptotic pathway, and mediate the pyroptosis.

Perfect cellular platform for electrophysiology:acutely isolated vagal ganglion neurons from adult canines / 中国药理学与毒理学杂志

OBJECTIVE To establish a platform of electrophysiology using vagal ganglion neurons (VGNs) isolated from adult canines. METHODS The VGNs were enzymatically isolated from adult canines of either gender and cultured under experimental conditions. Action potential (AP), repetitive firing, voltage-gated outward K+ currents (IK) and hyperpolarization-mediated inward currents (Ih) were recorded under current-and voltage-clamp configurations before and after treatment. RESULTS Analysis of AP waveform showed that ① inaddition to traditionally classified myelinated A- and unmyelinated C-types, myelinated Ah-types could also be identified in females rather than in males; ② step current depolarization evoked a stimulus intensity-dependent repetitive discharge, and to reach a similar firing frequency, the lowest stimulus intensity was required for A-types, a similar or slightly higher stimulus intensity was needed for Ah-types, and the highest stimulus intensity was required for C-types;③tetro?dotoxin significantly reduced the rate of depolarization and positively shifted the AP firing threshold of Ah-types, and iberiotoxin dramatically increased the neuroexcitability of Ah-types;④all tested neurons functionally expressed IK and Ih, and the current density for both channels on average was A-types>Ah-types>C-types; ⑤ although the distribution of afferent types of VGNs differed between males and females, the known difference in discharge profiles of A- and C-types isolated from male and female rats was not studied here. CONCLUSION The VGNs can be successfully isolated from adult canines, AP, IK and Ih can be recorded. The tight seal can be held for at least 30 min, which may be enough for pharmacological investigation.

Morphology and electrophysiological properties of neurons in the inner nuclear layer of the rat retina slices prepared by reformed method / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM: To investigate the morphology and electrophysiological properties of neurons in the inner nuclear layer(INL)of the adult rat retina slices. The retinal slices were prepared by low-melt agarose embedding and then cutted by vibratome. <p>METHODS: Whole cell patch clamp and intracellular staining with Lucifer Yellow were used in this study to study the morphology and electrophysiological properties of the INL neurons in retinal slices.<p>RESULTS: Retinal slices prepared in this method possessed a very smooth surface. The cells on the retinal slices maitained very good vitality, and some of the cells even retained their dendritic connections with other cells on the slice. According to the size and location of the cell bodies, neurons in the INL were easy to differentiate. Luciifer Yellow contained in the intracellular solution revealed the morphology of the recorded cell very well. Bipolar cells possessed elongated cell bodies and their processes mainly extended along the vertical direction. Horizontal cells and amacrine cells possessed much bigger and round cell bodies, resided in the outermost and inner most of the INL, respectively. The rest membrane potential and membrane capacitance of horizontal cell and amacrine cell were much higher than that of bipolar cells. Under a voltage step from -60mV to +40mV, 10mV per step, 41.7% of the cone bipolar cells and 64.7% of the amacrine cells exhibited inward sodium current and outward potassium current. Other cells only possessed outward potassium current. <p>CONCLUSION: The method of preparing retinal slices was very simple, and the viability of the slices were stable. These facilitated the patch-clamp recording of all the neurons in the INL including horizontal cells. Further investigation of the electrophysiological properties of the neurons in the INL was essential in revealing the mechanism of vision.

Etomidate reduces excitability of the neurons and suppresses the function of nAChR ventral horn in the spinal cord of neonatal rats / 南方医科大学学报

OBJECTIVE@#To investigate the effects of etomidate on electrophysiological properties and nicotinic acetylcholine receptors (nAChRs) of ventral horn neurons in the spinal cord.@*METHODS@#The spinal cord containing lumbosacral enlargement was isolated from 19 neonatal SD rats aged 7-12 days. The spinal cord were sliced and digested with papain (0.18 g/30 mL artificial cerebrospinal fluid) and incubated for 40 min. At the ventral horn, acute mechanical separation of neurons was performed with fire-polished Pasteur pipettes, and perforated patch-clamp recordings combined with pharmacological methods were employed on the adherent healthy neurons. In current-clamp mode, the spontaneous action potential (AP) of the ventral horn neurons in the spinal cord was recorded. The effects of pretreatment with different concentrations of etomidate on AP recorded in the ventral horn neurons were examined. In the voltage-clamp mode, nicotine was applied to induce inward currents in the ventral horn neurons, and the effect of pretreatment with etomidate on the inward currents induced by nicotine were examined with different etomidate concentrations, different holding potentials and different use time.@*RESULTS@#The isolated ventral horn neurons were in good condition with large diverse somata and intact processes. The isolated spinal ventral horn neurons (=21) had spontaneous action potentials, and were continuously perfused for 2 min with 0.3, 3.0 and 30.0 μmol/L etomidate. Compared with those before administration, the AP amplitude, spike potential amplitude and overshoot were concentration-dependently suppressed ( < 0.01), and spontaneous discharge frequency was obviously reduced ( < 0.01, =12). The APs of the other 9 neurons were completely abolished by etomidate at 3.0 or 30 μmol/L. At the same holding potential (VH=-70 mV), pretreatment with 0.3, 3.0 or 30.0 μmol/L etomidate for 2 min concentration-dependently suppressed the current amplitude induced by 0.4 mmol/L nicotine ( < 0.01, =7). At the holding potentials of - 30, - 50, and - 70 mV, pretreatment with 30.0 μmol/L etomidate for 2 min voltage-dependently suppressed the current amplitude induced by 0.4 mmol/L nicotine ( < 0.01, =6 for each holding potential). During the 6 min of 30.0 μmol/L etomidate pretreatment, the clamped cells were exposed to 0.4 mmol/L nicotine for 4 times at 0, 2, 4, and 6 min (each exposure time was 2 s), and the nicotinic current amplitude decreased gradually as the number of exposures increased. But at the same concentration, two nicotine exposures (one at the beginning and the other at the end of the 6 min pretreatment) resulted in a significantly lower inhibition rate compared with 4 nicotine exposures ( < 0.01, =6).@*CONCLUSIONS@#etomidate reduces the excitability of the spinal ventral neurons in a concentration-dependent manner and suppresses the function of nAChR in a concentration-, voltage-, and use-dependent manner.

Modulation of Dopaminergic Neuronal Excitability by Zinc through the Regulation of Calcium-related Channels

Depending on the intracellular buffering of calcium by chelation, zinc has the following two apparent effects on neuronal excitability: enhancement or reduction. Zinc increased tonic activity in the depolarized state when neurons were intracellularly dialyzed with EGTA but attenuated the neuronal activity when BAPTA was used as an intracellular calcium buffer. This suggests that neuronal excitability can be modulated by zinc, depending on the internal calcium buffering capacity. In this study, we elucidated the mechanisms of zinc-mediated alterations in neuronal excitability and determined the effect of calcium-related channels on zinc-mediated alterations in excitability. The zinc-induced augmentation of firing activity was mediated via the inhibition of small-conductance calcium-activated potassium (SK) channels with not only the contribution of voltage-gated L-type calcium channels (VGCCs) and ryanodine receptors (RyRs), but also through the activation of VGCCs via melastatin-like transient receptor potential channels. We suggest that zinc modulates the dopaminergic neuronal activity by regulating not only SK channels as calcium sensors, but also VGCCs or RyRs as calcium sources. Our results suggest that the cytosolic calcium-buffering capacity can tightly regulate zinc-induced neuronal firing patterns and that local calcium-signaling domains can determine the physiological and pathological state of synaptic activity in the dopaminergic system.

Selective serotonin reuptake inhibitor escitalopram inhibits 5-HT₃ receptor currents in NCB-20 cells

Escitalopram is one of selective serotonin reuptake inhibitor antidepressants. As an S-enantiomer of citalopram, it shows better therapeutic outcome in depression and anxiety disorder treatment because it has higher selectivity for serotonin reuptake transporter than citalopram. The objective of this study was to determine the direct inhibitory effect of escitalopram on 5-hydroxytryptamine type 3 (5-HT₃) receptor currents and study its blocking mechanism to explore additional pharmacological effects of escitalopram through 5-HT₃ receptors. Using a whole-cell voltage clamp method, we recorded currents of 5-HT₃ receptors when 5-HT was applied alone or co-applied with escitalopram in cultured NCB-20 neuroblastoma cells known to express 5-HT₃ receptors. 5-HT induced currents were inhibited by escitalopram in a concentration-dependent manner. EC50 of 5-HT on 5-HT₃ receptor currents was increased by escitalopram while the maximal peak amplitude was reduced by escitalopram. The inhibitory effect of escitalopram was voltage independent. Escitalopram worked more effectively when it was co-applied with 5-HT than pre-application of escitalopram. Moreover, escitalopram showed fast association and dissociation to the open state of 5-HT₃ receptor channel with accelerating receptor desensitization. Although escitalopram accelerated 5-HT₃ receptor desensitization, it did not change the time course of desensitization recovery. These results suggest that escitalopram can inhibit 5-HT₃ receptor currents in a non-competitive manner with the mechanism of open channel blocking.

Gastroprokinetic agent, mosapride inhibits 5-HT₃ receptor currents in NCB-20 cells

Mosapride accelerates gastric emptying by acting on 5-hydroxytryptamine type 4 (5-HT₄) receptor and is frequently used in the treatment of gastrointestinal (GI) disorders requiring gastroprokinetic efficacy. We tested the effect of mosapride on 5-hydroxytryptamine type 3 (5-HT₃) receptor currents because the 5-HT₃ receptors are also known to be expressed in the GI system and have an important role in the regulation of GI functions. Using the whole-cell voltage clamp method, we compared the currents of the 5-HT₃ receptors when 5-HT was applied alone or was co-applied with mosapride in cultured NCB-20 cells known to express 5-HT₃ receptors. The 5-HT₃ receptor current amplitudes were inhibited by mosapride in a concentration-dependent manner. Mosapride blocked the peak currents evoked by the application of 5-HT in a competitive manner because the EC₅₀ shifted to the right without changing the maximal effect. The rise slopes of 5-HT₃ receptor currents were decreased by mosapride. Pre-application of mosapride before co-application, augmented the inhibitory effect of mosapride, which suggests a closed channel blocking mechanism. Mosapride also blocked the opened 5-HT₃ receptor because it inhibited the 5-HT₃ receptor current in the middle of the application of 5-HT. It accelerated desensitization of the 5-HT₃ receptor but did not change the recovery process from the receptor desensitization. There were no voltage-, or use-dependency in its blocking effects. These results suggest that mosapride inhibited the 5-HT₃ receptor through a competitive blocking mechanism probably by binding to the receptor in closed state, which could be involved in the pharmacological effects of mosapride to treat GI disorders.