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patch clamp - Top 30 Publications

Examining the effects of alcohol on GABAA receptor mRNA expression and function in neural cultures generated from control and alcohol dependent donor induced pluripotent stem cells.

Factors influencing the development of alcohol-use disorder (AUD) are complex and heterogeneous. While animal models have been crucial to identifying actions of alcohol on neural cells, human-derived in vitro systems that reflect an individual's genetic background hold promise in furthering our understanding of the molecular and functional effects of alcohol exposure and the pathophysiology of AUD. In this report, we utilized induced pluripotent stem cell (iPSCs)-derived neural cell cultures obtained from healthy individuals (CTLs) and those with alcohol dependence (ADs) to 1) examine the effect of 21-day alcohol exposure on mRNA expression of three genes encoding GABAA receptor subunits (GABRA1, GABRG2, and GABRD) using quantitative PCR, and 2) examine the effect of acute and chronic alcohol exposure on GABA-evoked currents using whole-cell patch-clamp electrophysiology. iPSCs from CTLs and ADs were differentiated into neural cultures enriched for forebrain-type excitatory glutamate neurons. Following 21-day alcohol exposure, significant treatment effects were observed in GABRA1, GABRG2, and GABRD mRNA expression. A modestly significant interaction between treatment and donor phenotype was observed for GABRD, which was increased in cell cultures derived from ADs. No effect of acute or chronic alcohol was observed on GABA-evoked currents in neurons from either CTLs or ADs. This work extends findings examining the effects of alcohol on the GABAA receptor in human cell in vitro model systems.

Slice Patch Clamp Technique for Analyzing Learning-Induced Plasticity.

The slice patch clamp technique is a powerful tool for investigating learning-induced neural plasticity in specific brain regions. To analyze motor-learning induced plasticity, we trained rats using an accelerated rotor rod task. Rats performed the task 10 times at 30-s intervals for 1 or 2 days. Performance was significantly improved on the training days compared to the first trial. We then prepared acute brain slices of the primary motor cortex (M1) in untrained and trained rats. Current-clamp analysis showed dynamic changes in resting membrane potential, spike threshold, afterhyperpolarization, and membrane resistance in layer II/III pyramidal neurons. Current injection induced many more spikes in 2-day trained rats than in untrained controls. To analyze contextual-learning induced plasticity, we trained rats using an inhibitory avoidance (IA) task. After experiencing foot-shock in the dark side of a box, the rats learned to avoid it, staying in the lighted side. We prepared acute hippocampal slices from untrained, IA-trained, unpaired, and walk-through rats. Voltage-clamp analysis was used to sequentially record miniature excitatory and inhibitory postsynaptic currents (mEPSCs and mIPSCs) from the same CA1 neuron. We found different mean mEPSC and mIPSC amplitudes in each CA1 neuron, suggesting that each neuron had different postsynaptic strengths at its excitatory and inhibitory synapses. Moreover, compared with untrained controls, IA-trained rats had higher mEPSC and mIPSC amplitudes, with broad diversity. These results suggested that contextual learning creates postsynaptic diversity in both excitatory and inhibitory synapses at each CA1 neuron. AMPA or GABAA receptors seemed to mediate the postsynaptic currents, since bath treatment with CNQX or bicuculline blocked the mEPSC or mIPSC events, respectively. This technique can be used to study different types of learning in other regions, such as the sensory cortex and amygdala.

GI-530159, a novel, selective, mechano-sensitive K2P channel opener, reduces rat dorsal root ganglion (DRG) neuron excitability.

TREK two pore domain potassium channels play a critical role in regulating the excitability of somatosensory nociceptive neurons and are important mediators of pain perception. An understanding of the roles of TREK channels in pain perception and, indeed, in other pathophysiological conditions, has been severely hampered by the lack of potent and/or selective activators and inhibitors. In this study we describe a new, selective opener of TREK channels, GI-530159 (4,4'-(Hexafluoroisopropylidene)bis(p-phenyleneoxy)dianiline).

Is more always better? How different 'doses' of exercise after incomplete spinal cord injury affects the membrane properties of deep dorsal horn interneurons.

Interneurons in the deep dorsal horn (DDH) of the spinal cord process somatosensory input, and form an important link between upper and lower motoneurons to subsequently shape motor output. Exercise training after SCI is known to improve functional motor recovery, but little is known about the mechanisms within spinal cord neurons that underlie these improvements. Here we investigate how the properties of DDH interneurons are affected by spinal cord injury (SCI) alone, and SCI in combination with different 'doses' of treadmill exercise training (3, 6, and 9wks). In an adult mouse hemisection model of SCI we used whole-cell patch-clamp electrophysiology to record intrinsic, AP firing and gain modulation properties from DDH interneurons in a horizontal spinal cord slice preparation. We find that neurons within two segments of the injury, both ipsi- and contralateral to the hemisection, are similarly affected by SCI and SCI plus exercise. The passive intrinsic membrane properties input resistance (Rin) and rheobase are sensitive to the effects of recovery time and exercise training after SCI thus altering DDH interneuron excitability. Conversely, select active membrane properties are largely unaffected by either SCI or exercise training. SCI itself causes a mismatch in the expression of voltage-gated subthreshold currents and AP discharge firing type. Over time after SCI, and especially with exercise training (9wks), this mismatched expression is exacerbated. Lastly, amplification properties (i.e. gain of frequency-current relationship) of DDH interneurons are altered by SCI alone and recover spontaneously with no clear effect of exercise training. These results suggest a larger 'dose' of exercise training (9wks) has a strong and selective effect on specific membrane properties, and on the output of interneurons in the vicinity of a SCI. These electrophysiological data provide new insights into the plasticity of DDH interneurons and the mechanisms by which exercise therapy after SCI can improve recovery.

Novel intracellular transport-refractory mutations in KCNH2 identified in patients with symptomatic long QT syndrome.

Missense mutations in KCNH2, a gene encoding the Kv11.1 channel, cause long QT syndrome (LQTS) type 2 primarily by disrupting the intracellular transport of Kv11.1 to the plasma membrane. The present study aimed to clarify the functional changes by two novel KCNH2 missense mutations.

Drug interaction at hERG channel: In vitro assessment of the electrophysiological consequences of drug combinations and comparison against theoretical models.

Drugs carry a proarrhythmic risk, which gets even greater when they are used in combination. In vitro assessment of the proarrhythmic potential of drugs is limited to one compound and thus neglects the potential of drug-drug interactions, including those involving active metabolites. Here we present the results of an in vitro study of potential drug-drug interactions at the level of the hERG channel for the combination of up to three compounds: loratadine, desloratadine and ketoconazole. Experiments were performed at room temperature on an automated patch-clamp device CytoPatch 2, with the use of heterogeneously, stably transfected HEK cells. Single drugs, pairs and triplets were used. The results provided as the inhibition of the IKr current for pairs were compared against the calculated theoretical interaction. Models applied to calculate the combined effect of inhibitory actions of simultaneously given drugs include: (1) simple additive model with a maximal inhibition limit of 1 (all channels blocked in 100%); (2) Bliss independence; and (3) Loewe additivity. The observed IC50 values for loratadine, desloratadine and ketoconazole were 5.15, 1.95 and 0.74 μm respectively. For the combination of drugs tested in pairs, the effect was concentration dependent. In lower concentrations, the synergistic effect was observed, while for the highest tested concentrations it was subadditive. To triple the effect, it was subadditive regardless of concentrations. The square root of sum of squares of differences between the observed and predicted total inhibition was calculated to assess the theoretical interaction models. For most of the drugs, the allotopic model offered the best fit.

Short-term high-fat diet primes excitatory synapses for long-term depression in orexin neurons.

Overconsumption of high-fat diets is one of the strongest contributing factors to the rise of obesity rates. Orexin neurons are known to be activated by palatable high-fat diet and mediate the activation of the mesolimbic reward pathway, resulting in further food intake. While short-term exposure to high-fat diet is known to induce synaptic plasticity within the mesolimbic pathway, it is unknown if such changes occur in orexin neurons. To investigate this, 3-week old male rats were fed a palatable high-fat western diet (WD) or control chow for 1 week and then in vitro patch clamp recording was performed. In the WD condition, an activity-dependent long-term depression (LTD) of excitatory synapses was observed in orexin neurons, but not in chow controls. This LTD was presynaptic and depended on postsynaptic metabotropic glutamate receptor 5 (mGluR5) and retrograde endocannabinoid signalling. WD also increased extracellular glutamate levels, suggesting that glutamate spillover and subsequent activation of perisynaptic mGluR5 may occur more readily in the WD condition. In support of this, pharmacological inhibition of glutamate uptake was sufficient to prime chow control synapses to undergo a presynaptic LTD. Interestingly, these WD effects are transient, as extracellular glutamate levels were similar to controls and LTD was no longer observed in orexin neurons after 4 weeks of WD. In summary, excitatory synapses to orexin neurons become amenable to LTD under palatable high-fat diet, which may represent a homeostatic mechanism to prevent overactivation of these neurons and to curtail high-fat diet consumption. This article is protected by copyright. All rights reserved.

Developmental changes in spinal neuronal properties, motor network configuration and neuromodulation at free-swimming stages of Xenopus frog tadpoles.

We describe a novel preparation of the isolated brainstem and spinal cord from pro-metamorphic tadpole stages of the South African clawed frog (Xenopus laevis) that permits whole cell patch-clamp recordings from neurons in the ventral spinal cord. Previous research on earlier stages of the same species has provided one of the most detailed understandings of the design and operation of a CPG circuit. Here we have addressed how development sculpts complexity from this more basic circuit. The preparation generates bouts of fictive swimming activity either spontaneously or in response to electrical stimulation of the optic tectum, allowing an investigation into how the neuronal properties, activity patterns and neuromodulation of locomotor rhythm generation change during development. We describe an increased repertoire of cellular responses compared to younger larval stages and investigate the cellular level effects of nitrergic neuromodulation as well as the development of a sodium pump-mediated ultra-slow afterhyperpolarisation (usAHP) in these free-swimming larval animals.

TRPA1 and TRPV1 are required for lidocaine-evoked calcium influx and neuropeptide release but not cytotoxicity in mouse sensory neurons.

Local anaesthetics (LA) reduce neuronal excitability by inhibiting voltage-gated Na+ channels. When applied at high concentrations in the direct vicinity of nerves, LAs can also induce relevant irritation and neurotoxicity via mechanisms involving an increase of intracellular Ca2+. In the present study we explored the role of the Ca2+-permeable ion channels TRPA1 and TRPV1 for lidocaine-induced Ca2+-influx, neuropeptide release and neurotoxicity in mouse sensory neurons.

Human dental stem cell derived transgene-free iPSCs generate functional neurons via embryoid body-mediated and direct induction methods.

Induced pluripotent stem cells (iPSCs) give rise to neural stem/progenitor cells (NSCs), serving as a good source for neural regeneration. Here, we established transgene-free (TF) iPSCs from dental stem cells (DSCs) and determined their capacity to differentiate into functional neurons in vitro. Generated TF iPSCs from stem cells of apical papilla (SCAP) and dental pulp stem cells (DPSCs) underwent two methods -- embryoid body (EB)-mediated and direct induction, to guide TF-DSC iPSCs along with H9 or H9 Syn-GFP (human embryonic stem cells) into functional neurons in vitro. Using the EB-mediated method, early stage neural markers PAX6, SOX1 and nestin, were detected by immunocytofluorescence or RT-qPCR. At late stage of neural induction measured at weeks 7 and 9, the expression levels of neuron-specific markers Nav1.6, Kv1.4, Kv4.2, synapsin, SNAP25, PSD95, GAD67, GAP43 and NSE varied between SCAP iPSCs and H9. For direct induction method, iPSCs were directly induced into NSCs and guided to become neuron-like cells. The direct method while simpler, showed cell detachment and death during the differentiation process. At early stage, PAX6 and nestin were detected, whereas SOX1 was not. At late stage of differentiation, all 5 genes tested, nestin, βIII-tubulin, NFM, GFAP and NaV were positive in many cells in cultures. Both differentiation methods led to neuron-like cells in cultures exhibiting sodium and potassium currents, action potential or spontaneous excitatory postsynaptic potential. Thus, TF-DSC iPSCs are capable of undergoing guided neurogenic differentiation into functional neurons in vitro, thereby may serve as a cell source for neural regeneration.

Elevated potassium outward currents in hyperoxia treated atrial cardiomyocytes.

Supplementation of 100% oxygen is a very common intervention in intensive care units (ICU) and critical care centers for patients with dysfunctional lung and lung disorders. Although there is advantage in delivering sufficient levels of oxygen, hyperoxia is reported to be directly associated with increasing in-hospital deaths. Our previous studies reported ventricular and electrical remodeling in hyperoxia treated mouse hearts, and in this article, for the first time, we are investigating the effects of hyperoxia on atrial electrophysiology using whole-cell patch-clamp electrophysiology experiments along with assessment of Kv1.5, Kv4.2 and KChIP2 transcripts and protein profiles using real-time quantitative RT-PCR and Western blotting. Our data showed that induction of hyperoxia for 3 days in mice showed larger outward potassium currents with shorter action potential durations (APD). This increase in current densities is due to significant increase in ultrarapid delayed rectifier outward K(+) currents (IKur ) and rapidly activating, rapidly inactivating transient outward K+ current (Ito ) densities. We also observed a significant increase in both transcripts and protein levels of Kv1.5 and KChIP2 in hyperoxia treated atrial cardiomyocytes, whereas no significant change was observed in Kv4.2 transcripts or protein. The data presented here further support our previous findings that hyperoxia induces not only ventricular remodeling, but also atrial electrical remodeling. This article is protected by copyright. All rights reserved.

Effect of Low-Frequency Electrical Stimulation on the High-K(+)-Induced Neuronal Hyperexcitability in Rat Hippocampal Slices.

Low-frequency electrical stimulation (LFS) is a potential therapeutic method for epilepsy treatment. However, the effect of different LFS characteristics including the number of pulses, intensity and the time of application on its antiepileptic action has not been completely determined. In the present study, epileptiform activity (EA) was induced in hippocampal slices by high-K(+) solution which was washed out after 20 min. The changes in the electrophysiological properties of CA1 pyramidal neurons were measured during and 30 min after EA using whole-cell patch-clamp recording. EA occurrence resulted in neuronal hyperexcitability. Application of 1-Hz LFS to the Schaffer collaterals at 600 and 900 pulses and two intensities (equal and 1.5 times more than an intensity sufficient to elicits a 5-mV EPSP) at the beginning of EA showed that 900-pulse LFS at high intensity had stronger preventing effect on high-K(+)-induced neuronal hyperexcitability by increasing the rheobase current, utilization time, first-spike latency, delay to first-rebound action potential and decreasing the number of rebound action potential. In addition, application of high-intensity 900-pulse LFS had better inhibitory effect on the neuronal hyperexcitability when applied at the beginning of EA compared to its administration before or at 8-10 min after EA. Therefore, it may suggest the inhibitory action of LFS on the neuronal hyperexcitability is augmented by increasing its number of pulses and intensity. In addition, there is a time window for LFS application so that its application at the beginning of EA has better inhibitory effect.

Defective synaptic transmission causes disease signs in a mouse model of juvenile neuronal ceroid lipofuscinosis.

Juvenile neuronal ceroid lipofuscinosis (JNCL or Batten disease) caused by mutations in the CLN3 gene is the most prevalent inherited neurodegenerative disease in childhood resulting in widespread central nervous system dysfunction and premature death. The consequences of CLN3 mutation on the progression of the disease, on neuronal transmission, and on central nervous network dysfunction are poorly understood. We used Cln3 knockout (Cln3(Δex1-6)) mice and found increased anxiety-related behavior and impaired aversive learning as well as markedly affected motor function including disordered coordination. Patch-clamp and loose-patch recordings revealed severely affected inhibitory and excitatory synaptic transmission in the amygdala, hippocampus, and cerebellar networks. Changes in presynaptic release properties may result from dysfunction of CLN3 protein. Furthermore, loss of calbindin, neuropeptide Y, parvalbumin, and GAD65-positive interneurons in central networks collectively support the hypothesis that degeneration of GABAergic interneurons may be the cause of supraspinal GABAergic disinhibition.

Inhibition of the Voltage-Dependent K(+) Current by the Tricyclic Antidepressant Desipramine in Rabbit Coronary Arterial Smooth Muscle Cells.

We describe the effect of a tricyclic antidepressant drug desipramine on voltage-dependent K(+) (Kv) currents in freshly isolated rabbit coronary arterial smooth muscle cells using a conventional whole-cell patch clamp technique. Application of desipramine rapidly decreased the Kv current amplitude in a concentration-dependent manner, with an IC50 value of 5.91 ± 0.18 μM and a Hill coefficient of 0.61 ± 0.09. The steady-state inactivation curves of the Kv channels were not affected by desipramine. However, desipramine shifted the steady-state inactivation curves toward a more negative potential. Application of train pulses (1 or 2 Hz) slightly reduced the Kv current amplitude. Such reduction in the Kv current amplitude by train pulses increased in the presence of desipramine. Furthermore, the inactivation recovery time constant was also increased in the presence of desipramine, suggesting that desipramine-induced inhibition of the Kv current was use-dependent. Application of a Kv1.5 inhibitor (DPO-1) and/or a Kv2.1 inhibitor (guangxitoxin) did not change the inhibitory effect of desipramine on Kv currents. Based on these results, we concluded that desipramine directly inhibited the Kv channels in a dose- and state-dependent manner, but the effect was independent of norepinephrine/serotonin reuptake inhibition.

Dietary K(+) and Cl(-) independently regulate basolateral conductance in principal and intercalated cells of the collecting duct.

The renal collecting duct contains two distinct cell types, principal and intercalated cells, expressing potassium Kir4.1/5.1 (KCNJ10/16) and chloride ClC-K2 (ClC-Kb in humans) channels on their basolateral membrane, respectively. Both channels are thought to play important roles in controlling systemic water-electrolyte balance and blood pressure. However, little is known about mechanisms regulating activity of Kir4.1/5.1 and ClC-K2/b. Here, we employed patch clamp analysis at the single channel and whole cell levels in freshly isolated mouse collecting ducts to investigate regulation of Kir4.1/5.1 and ClC-K2/b by dietary K(+) and Cl(-) intake. Treatment of mice with high K(+) and high Cl(-) diet (6% K(+), 5% Cl(-)) for 1 week significantly increased basolateral K(+)-selective current, single channel Kir4.1/5.1 activity and induced hyperpolarization of basolateral membrane potential in principal cells when compared to values in mice on a regular diet (0.9% K(+), 0.5% Cl(-)). In contrast, basolateral Cl(-)-selective current and single channel ClC-K2/b activity was markedly decreased in intercalated cells under this condition. Substitution of dietary K(+) to Na(+) in the presence of high Cl(-) exerted a similar inhibiting action of ClC-K2/b suggesting that the channel is sensitive to variations in dietary Cl(-) per se. Cl(-)-sensitive with-no-lysine kinase (WNK) cascade has been recently proposed to orchestrate electrolyte transport in the distal tubule during variations of dietary K(+). However, co-expression of WNK1 or its major downstream effector Ste20-related proline-alanine-rich kinase (SPAK) had no effect on ClC-Kb over-expressed in Chinese hamster ovary (CHO) cells. Treatment of mice with high K(+) diet without concomitant elevations in dietary Cl(-) (6% K(+), 0.5% Cl(-)) elicited a comparable increase in basolateral K(+)-selective current, single channel Kir4.1/5.1 activity in principal cells, but had no significant effect on ClC-K2/b activity in intercalated cells. Furthermore, stimulation of aldosterone signaling by Deoxycorticosterone acetate (DOCA) recapitulated the stimulatory actions of high K(+) intake on Kir4.1/5.1 channels in principal cells but was ineffective to alter ClC-K2/b activity and basolateral Cl(-) conductance in intercalated cells. In summary, we report that variations of dietary K(+) and Cl(-) independently regulate basolateral potassium and chloride conductance in principal and intercalated cells. We propose that such discrete mechanism might contribute to fine-tuning of urinary excretion of electrolytes depending on dietary intake.

Effects of norquetiapine, the active metabolite of quetiapine, on cloned hERG potassium channels.

Quetiapine is an atypical antipsychotic drug that is widely used for the treatment of schizophrenia. It is mainly metabolized by a cytochrome P450 system in the liver. Norquetiapine is a major active metabolite in humans with a pharmacological profile that differs distinctly from that of quetiapine. We used the whole-cell patch-clamp technique to investigate the effects of norquetiapine on hERG channels that are stably expressed in HEK cells. Quetiapine and norquetiapine inhibited the hERG tail currents at -50mV in a concentration-dependent manner with IC50 values of 8.3 and 10.8μM, respectively, which suggested equal potency. The block of hERG currents by norquetiapine was voltage-dependent with a steep increase over a range of voltages for channel activation. However, at more depolarized potentials where the channels were fully activated, the block by norquetiapine was voltage-independent. The steady-state inactivation curve of the hERG currents was shifted to the hyperpolarizing direction in the presence of norquetiapine. Norquetiapine did not produce a use-dependent block. A fast application of norquetiapine inhibited the hERG current elicited by a 5s depolarizing pulse to +60mV, which fully inactivated the hERG currents, suggesting an inactivated-state block. During a repolarizing pulse wherein the hERG current was slowly deactivated, albeit remaining in an open state, a fast application of norquetiapine rapidly and reversibly inhibited the open state of the hERG current. Our results indicated that quetiapine and norquetiapine had equal potency in inhibiting hERG tail currents. Norquetiapine inhibited the hERG current by preferentially interacting with the open and/or inactivated states of the channels.

Suppressive effects of morphine injected into the ventral bed nucleus of the stria terminalis on the affective, but not sensory, component of pain in rats.

Pain is a complex experience with both sensory and affective components. Clinical and preclinical studies have shown that the affective component of pain can be reduced by doses of morphine lower than those necessary to reduce the sensory component. Although the neural mechanisms underlying the effects of morphine on the sensory component of pain have been investigated extensively, those influencing the affective component remain to be elucidated. The bed nucleus of the stria terminalis (BNST) has been implicated in the regulation of various negative emotional states, including aversion, anxiety, and fear. Thus, the present study aimed to clarify the role of the ventral part of the BNST (vBNST) in the actions of morphine on the affective and sensory components of pain. First, the effects of intra-vBNST injections of morphine on intraplantar formalin-induced conditioned place aversion (CPA) and nociceptive behaviors were investigated. Intra-vBNST injections of morphine reduced CPA without affecting nociceptive behaviors, which suggests that intra-vBNST morphine alters the affective, but not sensory, component of pain. Next, to examine the effects of morphine on neuronal excitability in type II vBNST neurons, whole-cell patch-clamp recordings were performed in brain slices. Bath application of morphine hyperpolarized type II vBNST neurons. Thus, the suppressive effects of intra-vBNST morphine on pain-induced aversion may be due to its inhibitory effects on neuronal excitability in type II vBNST neurons. These results suggest that the vBNST is a key brain region involved in the suppressive effects of morphine on the affective component of pain. This article is protected by copyright. All rights reserved.

Molecular function of α7 nicotinic receptors as drug targets.

Nicotinic acetylcholine receptors (nAChR) are pentameric ligand-gated ion channels involved in many physiological and pathological processes. In vertebrates, there are seventeen different nAChR subunits that combine to yield a variety of receptors with different pharmacology, function, and localization. The homomeric α7 receptor is one of the most abundant nAChRs in the nervous system and it is also present in non-neuronal cells. It plays important roles in cognition, memory, pain, neuroprotection, and inflammation. Its diverse physiological actions and associated disorders have made of α7 an attractive novel target for drug modulation. Potentiation of the α7 receptor has emerged as a novel therapeutic strategy for several neurological diseases, such as Alzheimer's and Parkinson's diseases, and inflammatory disorders. In contrast, increased α7 activity has been associated to cancer cell proliferation. The presence of different drug target sites offers a great potential for α7 modulation in different pathological contexts. In particular, compounds that target allosteric sites offer significant advantages over orthosteric agonists due to higher selectivity and a broader spectrum of degrees and mechanisms of modulation. Heterologous expression of α7, together with chaperone proteins, combined with patch clamp recordings have provided important advances in our knowledge of the molecular basis of α7 responses and their potential modulation for pathological processes. This review gives a synthetic view of α7 and its molecular function, focusing on how its unique activation and desensitization features can be modified by pharmacological agents. This fundamental information offers insights into therapeutic strategies. This article is protected by copyright. All rights reserved.

Kinetic Aspects of Verapamil Binding (On-Rate) on Wild-Type and Six hKv1.3 Mutant Channels.

The human-voltage gated Kv1.3 channel (hKv1.3) is expressed in T- and B lymphocytes. Verapamil is able to block hKv1.3 channels. We characterized the effect of verapamil on currents through hKv1.3 channels paying special attention to the on-rate (kon) of verapamil. By comparing on-rates obtained in wild-type (wt) and mutant channels a binding pocket for verapamil and impacts of different amino acid residues should be investigated.

Proteinase-Activated Receptor-2 Sensitivity of Amplified TRPA1 Activity in Skeletal Muscle Afferent Nerves and Exercise Pressor Reflex in Rats with Femoral Artery Occlusion.

Limb ischemia occurs in peripheral artery disease (PAD). Sympathetic nerve activity (SNA) that regulates blood flow directed to the ischemic limb is exaggerated during exercise in this disease, and transient receptor potential channel A1 (TRPA1) in thin-fiber muscle afferents contributes to the amplified sympathetic response. The purpose of the present study was to determine the role of proteinase-activated receptor-2 (PAR2) in regulating abnormal TRPA1 function and the TRPA1-mediated sympathetic component of the exercise pressor reflex.

Complex action of tyramine, tryptamine and histamine on native and recombinant ASICs.

Proton-gated channels of the ASIC family are widely distributed in the mammalian brain, and, according to the recent data, participate in synaptic transmission. However, ASIC-mediated currents are small, and special efforts are required to detect them. This prompts the search for endogenous ASIC ligands, which can activate or potentiate these channels. A recent finding of the potentiating action of histamine on recombinant homomeric ASIC1a has directed attention to amine-containing compounds. In the present study, we have analyzed the action of histamine, tyramine, and tryptamine on native and recombinant ASICs. None of the compounds caused potentiation of native ASICs in hippocampal interneurons. Furthermore, when applied simultaneously with channel activation, they produced voltage-dependent inhibition. Experiments on recombinant ASIC1a and ASIC2a allowed for an interpretation of these findings. Histamine and tyramine were found to be inactive on the ASIC2a, while tryptamine demonstrated weak inhibition. However, they induce both voltage-dependent inhibition of open channels and voltage-independent potentiation of closed/desensitized channels on the ASIC1a. We suggest that the presence of an ASIC2a subunit in heteromeric native ASICs prevents potentiation but not inhibition. As a result, the inhibitory action of histamine, which is masked by a strong potentiating effect on the ASIC1a homomers, becomes pronounced in experiments with native ASICs.

Angiotensin 1-7 Modulates Electrophysiological Characteristics and Calcium Homeostasis in Pulmonary Veins Cardiomyocytes via Mas/PI3K/eNOS signaling pathway.

Atrial fibrillation (AF) is the most common sustained arrhythmia, and pulmonary veins (PVs) play a critical role in triggering AF. Angiotensin (Ang)-(1-7) regulates calcium (Ca(2+) ) homeostasis and also plays a critical role in cardiovascular pathophysiology. However, the role of Ang-(1-7) in PV arrhythmogenesis remains unclear.

Comparison of 2D and 3D neural induction methods for the generation of neural progenitor cells from human induced pluripotent stem cells.

Neural progenitor cells (NPCs) from human induced pluripotent stem cells (hiPSCs) are frequently induced using 3D culture methodologies however, it is unknown whether spheroid-based (3D) neural induction is actually superior to monolayer (2D) neural induction. Our aim was to compare the efficiency of 2D induction with 3D induction method in their ability to generate NPCs, and subsequently neurons and astrocytes. Neural differentiation was analysed at the protein level qualitatively by immunocytochemistry and quantitatively by flow cytometry for NPC (SOX1, PAX6, NESTIN), neuronal (MAP2, TUBB3), cortical layer (TBR1, CUX1) and glial markers (SOX9, GFAP, AQP4). Electron microscopy demonstrated that both methods resulted in morphologically similar neural rosettes. However, quantification of NPCs derived from 3D neural induction exhibited an increase in the number of PAX6/NESTIN double positive cells and the derived neurons exhibited longer neurites. In contrast, 2D neural induction resulted in more SOX1 positive cells. While 2D monolayer induction resulted in slightly less mature neurons, at an early stage of differentiation, the patch clamp analysis failed to reveal any significant differences between the electrophysiological properties between the two induction methods. In conclusion, 3D neural induction increases the yield of PAX6(+)/NESTIN(+) cells and gives rise to neurons with longer neurites, which might be an advantage for the production of forebrain cortical neurons, highlighting the potential of 3D neural induction, independent of iPSCs' genetic background.

Phytochemicals genistein and capsaicin modulate Kv2.1 channel gating.

Phytochemicals are a large group of plant-derived compounds that have a broad range of pharmacological effects. Some of these effects are derived from their action on transport proteins, including ion channels. The present study investigates the effects of the phytochemicals genistein and capsaicin on voltage-gated potassium Kv2.1 channels.

Biophysical comparison of sodium currents in native cardiac myocytes and human induced pluripotent stem cell-derived cardiomyocytes.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are used for safety pharmacology and to investigate genetic diseases affecting cardiac ion channels. It is unclear whether adult myocytes or hiPSC-CMs are the better platform for cardiac safety pharmacology. We examined the biophysical and molecular properties of INa in adult myocytes and hiPSC-CMs.

Neuroprotective effect of cannabinoid receptor 1 antagonist in the MNU-induced retinal degeneration model.

Endocannabinoid system involves in neuroprotective effects on the central neural system. The cannabinoid receptor 1 (CB1R) is widely expressed in the mouse retina. However, the role of cannabinoid receptors in the retina remains unclear. In this work, we established a photoreceptor degeneration mouse model via N-methyl-N-nitrosourea (MNU) administration to identify the neuroprotective effects of cannabinoid receptors. The MNU-induced retinal degeneration behaves similarly to that in the human retinitis pigmentosa (RP). Administration of the CB1R antagonist SR141716A distinctly recovered the photoreceptor loss, decreased glial reactivity and reduced abnormal vascular complexes in an MNU-induced mouse model. The BC dendrites were shrunk in the MNU-treated retina with eliminated ON-BCs responses and partially diminished OFF-BCs responses in patch-clamp recordings. In the MNU + SR1 group, both the function and structure of ON-BCs recovered. Taken together, our study showed that the inhibition of CB1R can effectively prevent MNU-induced retinal degeneration, suggesting a potential therapeutic effect of the CB1R antagonist SR1 in retinal degeneration diseases.

Role of TRH/UCN3 neurons of the perifornical area/bed nucleus of stria terminalis region in the regulation of the anorexigenic POMC neurons of the arcuate nucleus in male mice and rats.

Two anorexigenic peptides, thyrotropin-releasing hormone (TRH) and urocortin 3 (UCN3), are co-expressed in a continuous neuronal group that extends from the perifornical area to the bed nucleus of stria terminalis, raising the possibility that this cell group may be involved in the regulation of energy homeostasis. In this study, therefore, we tested the hypothesis that the TRH/UCN3 neurons regulate food intake by influencing feeding-related neuropeptide Y (NPY) and/or proopiomelanocortin (POMC) neurons in the arcuate nucleus (ARC). Triple-labeled immunofluorescent preparations demonstrated that only very few NPY neurons (4.3 ± 1.3%) were contacted by double-labeled TRH/UCN3 axons in the ARC. In contrast, more than half of the POMC neurons (52.4 ± 8.5%) were contacted by double-labeled axons. Immuno-electron microscopy demonstrated that the UCN3 axons established asymmetric synapses with POMC neurons, indicating the excitatory nature of these synaptic specializations. Patch clamp electrophysiology revealed that TRH and UCN3 have antagonistic effects on the POMC neurons. While UCN3 depolarizes and increases the firing rate of POMC neurons, TRH prevents these effects of UCN3. These data demonstrate that TRH/UCN3 neurons in the perifornical/BNST region establish abundant synaptic associations with the POMC neurons in the ARC and suggest a potentially important role for these neurons in the regulation of food intake through an antagonistic interaction between TRH and UCN3 on the electrophysiological properties of POMC neurons.

MiR-335 overexpression impairs insulin secretion through defective priming of insulin vesicles.

MicroRNAs contribute to the maintenance of optimal cellular functions by fine-tuning protein expression levels. In the pancreatic β-cells, imbalances in the exocytotic machinery components lead to impaired insulin secretion and type 2 diabetes (T2D). We hypothesize that dysregulated miRNA expression exacerbates β-cell dysfunction, and have earlier shown that islets from the diabetic GK-rat model have increased expression of miRNAs, including miR-335-5p (miR-335). Here, we aim to determine the specific role of miR-335 during development of T2D, and the influence of this miRNA on glucose-stimulated insulin secretion and Ca(2+)-dependent exocytosis. We found that the expression of miR-335 negatively correlated with secretion index in human islets of individuals with prediabetes. Overexpression of miR-335 in human EndoC-βH1 and in rat INS-1 832/13 cells (OE335) resulted in decreased glucose-stimulated insulin secretion, and OE335 cells showed concomitant reduction in three exocytotic proteins: SNAP25, Syntaxin-binding protein 1 (STXBP1), and synaptotagmin 11 (SYT11). Single-cell capacitance measurements, complemented with TIRF microscopy of the granule marker NPY-mEGFP demonstrated a significant reduction in exocytosis in OE335 cells. The reduction was not associated with defective docking or decreased Ca(2+) current. More likely, it is a direct consequence of impaired priming of already docked granules. Earlier reports have proposed reduced granular priming as the cause of reduced first-phase insulin secretion during prediabetes. Here, we show a specific role of miR-335 in regulating insulin secretion during this transition period. Moreover, we can conclude that miR-335 has the capacity to modulate insulin secretion and Ca(2+)-dependent exocytosis through effects on granular priming.

Developmental refinement of synaptic transmission on micropatterned single layer graphene.

Interfacing neurons with graphene, a single atomic layer of sp(2) hybridized C-atoms, is a key paradigm in understanding how to exploit the unique properties of such a two-dimensional system for neural prosthetics and biosensors development. In order to fabricate graphene-based circuitry, a reliable large area patterning method is a requirement. Following a previously developed protocol, we monitored the in vitro neuronal development of geometrically ordered neural network growing onto patterned Single Layer Graphene (SLG) coated with poly-D-lysine. The microscale patterns were fabricated via laser micromachining and consisted of SLG stripes separated by micrometric ablated stripes. A comprehensive analysis of the biointerface was carried out combining the surface characterization of SLG transferred on the glass substrates and Immunohistochemical (IHC) staining of the developing neural network. Neuronal and glial cells proliferation, as well as cell viability, were compared on glass, SLG and SLG-patterned surfaces. Further, we present a comparative developmental study on the efficacy of synaptic transmission on control glass, on transferred SLG, and on the micropatterned SLG substrates by recording miniature post synaptic currents (mPSCs). The mPSC frequencies and amplitudes obtained on SLG-stripes, SLG only and on glass were compared. Our results indicate a very similar developmental trend in the three groups, indicating that both SLG and patterned SLG preserve synaptic efficacy and can be potentially exploited for the fabrication of large area devices for neuron sensing or stimulation.

Direct interaction with 14-3-3γ promotes surface expression of Best1 channel in astrocyte.

Bestrophin-1 (Best1) is a calcium-activated anion channel (CAAC) that is expressed broadly in mammalian tissues including the brain. We have previously reported that Best1 is expressed in hippocampal astrocytes at the distal peri-synaptic regions, called microdomains, right next to synaptic junctions, and that it disappears from the microdomains in Alzheimer's disease mouse model. Although Best1 appears to be dynamically regulated, the mechanism of its regulation and modulation is poorly understood. It has been reported that a regulatory protein, 14-3-3 affects the surface expression of numerous membrane proteins in mammalian cells.