Search

Introduction: Melanin-concentrating hormone (MCH) neurons are essential regulators of energy and glucose homeostasis, sleep–wake behaviors, motivation, learning and memory. These neurons are anatomically distributed across the medial (MH) and lateral hypothalamus (LH), and the adjacent zona incerta (ZI), which may represent functional subgroups with distinct connectivity with different brain regions. Furthermore, MCH neurons can be classified according to co-expression of neuropeptides, such as cocaine and amphetamine- regulated transcript (CART). Methods: To identify functional similarities and differences of MCH subpopulations, we characterized their intrinsic electrophysiological properties using whole cell current clamp recording on acute brain slices from male and female mice. Results: MCH neurons were classified into subgroups according to their anatomical localization in three MCH-rich brain areas: MH, LH and ZI. Among the three brain regions, ZI MCH neurons were the least excitable while LH MCH neurons were the most excitable. Furthermore, grouping MCH neurons according to CART co-expression revealed that MCH/CART− cells are uniquely depolarized and excitable, and display H-currents. These MCH/CART− cells were mainly found in the LH, which may in part explain why LH MCH neurons are more excitable. While some sex differences were found, the majority of parameters investigated were not different. Discussion: Our results suggest that MCH/CART− cells are electrophysiologically distinct, whereas MCH/CART+ cells are largely similar despite their diffuse distribution in the hypothalamus. It is therefore a combination of intrinsic electrophysiological properties and neurochemical identities, in addition to anatomy and connectivity that are likely to be critical in defining functional subpopulations of MCH neurons. [ABSTRACT FROM AUTHOR]

The Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative reassesses using the inhibition of hERG potassium channels by drugs as the major determinant for the potential to cause drug-induced Torsades de Pointes (TdP) cardiac arrhythmias. Here we report our findings on the next phase of CiPA: Determination of hERG inhibitory properties using the standard CiPA-defined data acquisition protocol, here called the standard protocol, at physiological temperature (37 degrees Celsius). To do this, we measured inhibition of hERG1a potassium channels stably expressed in HEK293 cells by the small molecule verapamil, using manual whole-cell patch-clamp electrophysiology recordings with the standard protocol, which is characterized, in part, by a series of 10 s duration voltage steps to 0 mV, ultimately leading to a cumulative recording time of approximately 30 min. Using the standard protocol, we measured an IC 50 for verapamil of 225 nM, a Hill coefficient of 1, and time constant of inhibition at 0 mV of 0.64 s. But, using the standard protocol resulted in a very low (5 %) experimental success rate per cell, which had low practicality for future experiments. To address the 5 % success rate, we generated a revised protocol characterized, in part, by a series of 3 s duration voltage steps to 0 mV, leading to a cumulative recording time of approximately 10 min. Using the revised protocol, we found an IC 50 for verapamil of 252 nM, a Hill coefficient of 0.8, and time constant of inhibition at 0 mV of 0.67 s. The values measured with the revised protocol were similar to those measured using the standard protocol and, furthermore, our success rate using the revised protocol rose to 25 %, an increase of 5-fold over the standard protocol, and more in line with the success rate for biophysical studies. In summary, we captured key pharmacological data for subsequent analysis in CiPA using a revised protocol with an increased success rate and an overall enhanced feasibility and practicality. We propose that the revised protocol may be more pragmatic for generation of some hERG channel drug inhibition data for CiPA and other regulatory sciences. [ABSTRACT FROM AUTHOR]

Key points: Carotid body (CB) chemoreceptors are hyperactive in hypertension, and their acute activation produces bronchoconstriction.We show that the respiratory‐modulated bronchiolar tone, pulmonary parasympathetic efferent activity, and the firing frequency and synaptic excitation of bronchoconstrictor motoneurones in the nucleus ambiguus were all enhanced in spontaneous hypertensive (SH) rats.In SH rats, CB denervation reduced the respiratory‐related parasympathetic‐mediated bronchoconstrictor tone to levels seen in normotensive rats.Chemoreflex evoked bronchoconstrictor tone was heightened in SH versus normotensive rats.The intrinsic electrophysiological properties and morphology of bronchoconstrictor motoneurones were similar across rat strains.The heightened respiratory modulation of parasympathetic‐mediated bronchoconstrictor tone to the airways in SH rats is caused by afferent drive from the CBs. Much research has described heightened sympathetic activity in hypertension and diminished parasympathetic tone, especially to the heart. The carotid body (CB) chemoreceptors exhibit hyperreflexia and are hyperactive, providing excitatory drive to sympathetic networks in hypertension. Given that acute CB activation produces reflex evoked bronchoconstriction via activation of parasympathetic vagal efferents, we hypothesised that the parasympathetic bronchoconstrictor activity is enhanced in spontaneously hypertensive (SH) rats and that this is dependent on CB inputs. In situ preparations of Wistar and SH rats were used in which bronchiolar tone, the pulmonary branch of the vagus (pVN) and phrenic nerves were recorded simultaneously; whole cell patch clamp recordings of bronchoconstrictor vagal motoneurones were also made from the nucleus ambiguus. Bronchiolar tone, pVN and bronchoconstrictor motoneurones were respiratory modulated and this modulation was enhanced in SH rats. These differences were all eliminated after CB denervation. Stimulation of the CBs increased the phrenic frequency that caused a summation of the respiratory‐related increases in pVN, resulting in the development of bronchoconstrictor tone. This tone was exaggerated in SH rats. The enhanced respiratory‐parasympathetic coupling to airways in SH rats was not due to differences in the intrinsic electrophysiological properties of bronchoconstrictor motoneurones but reflected heightened pre‐inspiratory‐ and inspiratory‐related synaptic drive. In summary, in SH rats the phasic respiratory modulation of parasympathetic tone to the airways is elevated and the greater development of this bronchoconstrictor tone is caused by the heightened afferent drive originating from the CBs. Thus, targeting the CBs may prove effective for increasing lower airway patency. Key points: Carotid body (CB) chemoreceptors are hyperactive in hypertension, and their acute activation produces bronchoconstriction.We show that the respiratory‐modulated bronchiolar tone, pulmonary parasympathetic efferent activity, and the firing frequency and synaptic excitation of bronchoconstrictor motoneurones in the nucleus ambiguus were all enhanced in spontaneous hypertensive (SH) rats.In SH rats, CB denervation reduced the respiratory‐related parasympathetic‐mediated bronchoconstrictor tone to levels seen in normotensive rats.Chemoreflex evoked bronchoconstrictor tone was heightened in SH versus normotensive rats.The intrinsic electrophysiological properties and morphology of bronchoconstrictor motoneurones were similar across rat strains.The heightened respiratory modulation of parasympathetic‐mediated bronchoconstrictor tone to the airways in SH rats is caused by afferent drive from the CBs. [ABSTRACT FROM AUTHOR]

T-type Ca channels are strongly expressed and important in the developing heart. In the adult heart, these channels play a significant role in pacemaker tissues, but there is uncertainty about their presence and physiological relevance in the working myocardium. Here, we show that the T-type Ca channel isoforms Cav3.1 and Cav3.2 are expressed at a protein level in ventricular cardiomyocytes from healthy adult C57/BL6 mice. Myocytes isolated from adult wild-type and Cav3.2 KO mice showed considerable whole cell T-type Ca currents under beta-adrenergic stimulation with isoprenaline. We further show that the detectability of basal T-type Ca currents in murine wild-type cardiomyocytes depends on the applied experimental conditions. Together, these findings reveal the presence of functional T-type Ca channels in the membrane of ventricular myocytes. In addition, electrically evoked Ca release from the sarcoplasmic reticulum was significantly impaired in Cav3.2 KO compared to wild-type cardiomyocytes. Our work implies a physiological role of T-type Ca channels in the healthy adult murine ventricular working myocardium.

Abstract Background High-grade chondrosarcoma, which has a high incidence of local recurrence and pulmonary metastasis despite surgical resection, is associated with poor prognosis. Therefore, new and effective adjuvant therapies are urgently required for this disease. Gamma-aminobutyric acid (GABA), which acts as a neurotrophic factor during nervous system development, is related to the proliferation and migration of certain cancer cells. The GABAergic system, which is composed of GABA, the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD), and GABA receptors, has an important function in nerve growth and development of neural crest. Therefore, the GABAergic system may play important functional roles in the proliferation of chondrosarcoma cells, which are derived from neural crest cells. We examined the anti-tumor effects of the GABAergic system on a chondrosarcoma cell line. Methods We evaluated the underlying mechanisms of the anti-tumor effects of the GABAergic system, such as the involvement of different signaling pathways, apoptosis, and cell cycle arrest, in the high-grade chondrosarcoma cell line OUMS-27. In addition, we performed whole-cell patch-clamp recordings for Ca2+ currents and evaluated the changes in intracellular Ca2+ concentration via Ca2+ channels, which are related to the GABAB receptor in high-grade chondrosarcoma cells. Results The GABAB receptor antagonist CGP had anti-tumor effects on high-grade chondrosarcoma cells in a dose-dependent manner. The activities of caspase 3 and caspase 9 were significantly elevated in CGP-treated cells compared to in untreated cells. The activity of caspase 8 did not differ significantly between untreated cells and CGP-treated cells. However, caspase 8 tended to be up-regulated in CGP-treated cells. The GABAB receptor antagonist exhibited anti-tumor effects at the G1/S cell cycle checkpoint and induced apoptosis via dual inhibition of the PI3/Akt/mTOR and MAPK signaling pathways. Furthermore, the changes in intracellular Ca2+ via GABAB receptor-related Ca2+ channels inhibited the proliferation of high-grade chondrosarcoma cells by inducing and modulating apoptotic pathways. Conclusions The GABAB receptor antagonist may improve the prognosis of high-grade chondrosarcoma by exerting anti-tumor effects via different signaling pathways, apoptosis, cell cycle arrest, and Ca2+ channels in high-grade chondrosarcoma cells.

Neuronal nitric oxide synthase (nNOS) is considered a regulator of Cav1.2 L-type Ca2+ channels and downstream Ca2+ cycling in the heart. The commonest view is that nitric oxide (NO), generated by nNOS activity in cardiomyocytes, reduces the currents through Cav1.2 channels. This gives rise to a diminished Ca2+ release from the sarcoplasmic reticulum, and finally reduced contractility. Here, we report that nNOS inhibitor substances significantly increase intracellular Ca2+ transients in ventricular cardiomyocytes derived from adult mouse and rat hearts. This is consistent with an inhibitory effect of nNOS/NO activity on Ca2+ cycling and contractility. Whole cell currents through L-type Ca2+ channels in rodent myocytes, on the other hand, were not substantially affected by the application of various NOS inhibitors, or application of a NO donor substance. Moreover, the presence of NO donors had no effect on the single-channel open probability of purified human Cav1.2 channel protein reconstituted in artificial liposomes. These results indicate that nNOS/NO activity does not directly modify Cav1.2 channel function. We conclude that—against the currently prevailing view—basal Cav1.2 channel activity in ventricular cardiomyocytes is not substantially regulated by nNOS activity and NO. Hence, nNOS/NO inhibition of Ca2+ cycling and contractility occurs independently of direct regulation of Cav1.2 channels by NO. [ABSTRACT FROM AUTHOR]

Parkinson’s disease (PD) is predominantly idiopathic in origin, and a large body of evidence indicates that gastrointestinal (GI) dysfunctions are a significant comorbid clinical feature; these dysfunctions include dysphagia, nausea, delayed gastric emptying, and severe constipation, all of which occur commonly before the onset of the well-known motor symptoms of PD. Based on a distinct distribution pattern of Lewy bodies (LB) in the enteric nervous system (ENS) and in the preganglionic neurons of the dorsal motor nucleus of the vagus (DMV), and together with the early onset of GI symptoms, it was suggested that idiopathic PD begins in the ENS and spreads to the central nervous system (CNS), reaching the DMV and the substantia nigra pars compacta (SNpc). These two areas are connected by a recently discovered monosynaptic nigro-vagal pathway, which is dysfunctional in rodent models of PD. An alternative hypothesis downplays the role of LB transport through the vagus nerve and proposes that PD pathology is governed by regional or cell-restricted factors as the leading cause of nigral neuronal degeneration. The purpose of this brief review is to summarize the neuronal electrophysiological findings in the SNpc and DMV in PD. [ABSTRACT FROM AUTHOR]

Objective: Resveratrol, a phytoalexin, has a wide range of desirable biological actions. Despite a growing body of evidence indicating that resveratrol induces changes in neuronal function, little effort, if any, has been made to investigate the cellular effect of resveratrol treatment on intrinsic neuronal properties. Materials and Methods: This experimental study was performed to examine the acute effects of resveratrol (100 μM) on the intrinsic evoked responses of rat Cornu Ammonis (CA1) pyramidal neurons in brain slices, using whole cell patch clamp recording under current clamp conditions. Results: Findings showed that resveratrol treatment caused dramatic changes in evoked responses of pyramidal neurons. Its treatment induced a significant (P

The effect of celecoxib on electrophysiological property of Nav1.5 was studied，which was associated with cardiac toxicity. The effects of celecoxib on the electrophysiological properties of Nav 1.5， including peak current， voltage-dependent activation， voltage-dependent inactivation and recovery kinetics， were investigated by whole cell patch clamp technique. The results showed that celecoxib inhibited the peak currents of Nav1.5 in a concentration-dependent manner with a calculated IC50 value of 1.54×10-8mol/L. Moreover， celecoxib promoted the activation and inactivation phase of Nav1.5， making it somewhat difficult to recover from the inactivation to the resting state. The prominent inhibition of celecoxib on Nav1.5 indicated that there might be a close relationship between potential cardiovascular risks and Nav1.5. [ABSTRACT FROM AUTHOR]

Nifedipine and isradipine are prominent examples of calcium channel blockers with a 1,4-dihydropyridine (DHP) scaffold. Although successfully used in clinics since decades for the treatment of hypertension, the binding mechanism to their target, the L-type voltage-gated calcium channel Cav1.2, is still incompletely understood. Recently, novel DHP derivatives with a condensed ring system have been discovered that show distinct selectivity profiles to different calcium channel subtypes. This property renders this DHP class as a promising tool to achieve selectivity towards distinct calcium channel subtypes. In this study, we identified a common binding mode for prominent DHPs nifedipine and isradipine using docking and pharmacophore analysis that is also able to explain the structure-activity relationship of a small subseries of DHP derivatives with a condensed ring system. These findings were used to guide the synthesis of twenty-two novel DHPs. An extensive characterization using 1 H NMR, 13 C NMR, mass spectra and elemental analysis was followed by whole cell patch clamp assays for analyzing activity at Cav1.2 and Cav3.2. Two compounds were identified with significant activity against Cav1.2. Additionally, we identified four compounds active against Cav3.2 of which three were selective over Cav1.2. Novel binding modes were analyzed using docking and pharmacophore analysis as well as molecular dynamics simulations. [ABSTRACT FROM AUTHOR]

Testosterone synthesis within Leydig cells is a calcium-dependent process. Intracellular calcium levels are regulated by different processes including ATP-activated P2X purinergic receptors, T-type Ca2+ channels modulated by the luteinizing hormone, and intracellular calcium storages recruited by a calcium-induced calcium release mechanism. On the other hand, nitric oxide (NO) is reported to have an inhibitory role in testosterone production. Based on these observations, we investigated the interaction between the purinergic and nitrergic systems in Leydig cells of adult mice. For this purpose, we recorded ATP-evoked currents in isolated Leydig cells using the whole cell patch clamp technique after treatment with L-NAME (300 μM and 1 mM), L-arginine (10, 100, 300, and 500 μM), ODQ (300 μM), and 8-Br-cGMP (100 μM). Our results show that NO produced by Leydig cells in basal conditions is insufficient to change the ATP-evoked currents and that extra NO provided by adding 300 μM L-arginine positively modulates the current through a mechanism involving the NO/cGMP signaling pathway. Thus, we report an interaction between the nitrergic and purinergic systems in Leydig cells and suggest that Ca2+ entry via the purinergic receptors can be regulated by NO.

Intrinsic neuronal excitability has been reported to change during normal aging. The bed nucleus of the stria terminalis (BNST), a limbic forebrain structure, is involved in fear, stress and anxiety; behavioral features that exhibit age-dependent properties. To examine the effect of aging on intrinsic neuronal properties in BNST we compared patch clamp recordings from cohorts of female mice at two ages, 3–4 months (Young) and 29–30 months (Aged) focusing on 2 types of BNST neurons. Aged Type I neurons exhibited a hyperpolarized resting membrane potential (RMP) of circa -80 mV compared to circa -70 mV in the Young. A key finding in this study is a hyper-excitability of Type II neurons with age reflected in an increase in firing frequency in response to depolarizing current injections; activation of Type II neurons is believed to dampen anxiety like responses. Such age-related changes in intrinsic neurophysiological function are likely to modulate how the limbic system, acting via BNST, shapes function in the HPA-axis.

Stellera chamaejasme L. (Thymelaeaceae) is a toxic perennial herb and widespread in Mongolia and the northern parts of China. Previous studies have revealed that Neochamaejasmin A (NCA), one of the main active ingredients in the plant roots, has many bioactivities such as inhibiting the P-gp-mediated efflux. But whether NCA affects ion channels is unknown. Here the whole cell patch clamp technique was used to investigate whether NCA affects ion channels, especially how it inhibits K V 1.4. Mutagenesis and structure-based molecular simulation were used for analysis of inhibition mechanism and identification of binding site. Among all the channels assayed, K V 1.4 stood out as the one on which NCA showed strongest inhibition activity with IC 50 of 7.55 µM. Compared with NCA’s isomerides, neochamaejasmin B (NCB) and chamaechromone (CMC), NCA also exhibited superior inhibition ability on K V 1.4. Three mutations, V549A, A553V and V560A, occurred inside the pore, were found to significantly alleviate the NCA blocking effects, suggesting that they are the important binding sites of NCA. Structure-based modelling showed that the phenolic hydroxyl group of NCA can form hydrogen bonds with main chains of Val549 and Ala553 in IS6 and IVS6 segment respectively, which support our in vitro results. In conclusion, data suggest that NCA might inhibit K V 1.4 channels via direct binding to the pore domain. [ABSTRACT FROM AUTHOR]

Intrinsic neuronal excitability has been reported to change during normal aging. The bed nucleus of the stria terminalis (BNST), a limbic forebrain structure, is involved in fear, stress and anxiety; behavioral features that exhibit age-dependent properties. To examine the effect of aging on intrinsic neuronal properties in BNST we compared patch clamp recordings from cohorts of female mice at two ages, 3-4 months (Young) and 29-30 months (Aged) focusing on 2 types of BNST neurons. Aged Type I neurons exhibited a hyperpolarized resting membrane potential (RMP) of circa -80 mV compared to circa -70 mV in the Young. A key finding in this study is a hyper-excitability of Type II neurons with age reflected in an increase in firing frequency in response to depolarizing current injections; activation of Type II neurons is believed to dampen anxiety like responses. Such age-related changes in intrinsic neurophysiological function are likely to modulate how the limbic system, acting via BNST, shapes function in the HPA-axis. [ABSTRACT FROM AUTHOR]

The ventral pallidum (VP) is an integral locus in the reward circuitry and a major target of GABAergic innervation of both D1-medium spiny neurons (MSNs) and D2-MSNs from the nucleus accumbens. The VP contains populations of GABAergic [VPGABA, GAD2(+), or VGluT(-)] and glutamatergic [VPGlutamate, GAD2(-), or VGluT(+)] cells that facilitate positive reinforcement and behavioral avoidance, respectively. MSN efferents to the VP exert opponent control over behavioral reinforcement with activation of D1-MSN afferents promoting and D2-MSN afferents inhibiting reward seeking. How this afferent-specific and cell type-specific control of reward seeking is integrated remains largely unknown. In addition to GABA, D1-MSNs corelease substance P to stimulate neurokinin 1 receptors (NK1Rs) and D2-MSNs corelease enkephalin to activate μ-opioid receptors (MORs) and δ-opioid receptors. These neuropeptides act in the VP to alter appetitive behavior and reward seeking. Using a combination of optogenetics and patch-clamp electrophysiology in mice, we found that GAD2(-) cells receive weaker GABA input from D1-MSN, but GAD2(+) cells receive comparable GABAergic input from both afferent types. Pharmacological activation of MORs induced an equally strong presynaptic inhibition of GABA and glutamate transmission on both cell types. Interestingly, MOR activation hyperpolarized VPGABA but not VGluT(+). NK1R activation inhibited glutamatergic transmission only on VGluT(+) cells. Our results indicate that the afferent-specific release of GABA and neuropeptides from D1-MSNs and D2-MSNs can differentially influence VP neuronal subtypes., (Copyright © 2023 Neuhofer and Kalivas.)

The mechanisms by which G proteins modulate voltage-gated Ca2+ channel currents (CaV), particularly CaV2.2 and CaV2.3, are voltage dependent (VD) or voltage independent (VI). VD pathways are typically mediated by Gαi/o and GαS subfamilies. On the other hand, VI inhibition modulation is coupled to the Gαq subfamily and signaling pathways downstream of phospholipase C stimulation. In most studies, this latter pathway has been shown to be linked to Gαq and/or Gα11 protein subunits. However, there are no studies that have examined whether natively expressed Gα14 subunits (Gαq subfamily member) couple G protein-coupled receptors (GPCR) with CaV2.2 channels. We report that Gα14 subunits functionally couple the substance P (SP)/neurokinin-1 (NK-1) receptor pathway to CaV2.2 channels in acutely dissociated rat celiac-superior mesenteric ganglion (CSMG) neurons. Exposure of CSMG neurons to SP blocked the CaV2.2 currents in a predominantly VD manner that was pertussis toxin and cholera toxin resistant, as well as Gαq/11 independent. However, silencing Gα14 subunits significantly attenuated the SP-mediated Ca2+ current block. In another set of experiments, exposure of CSMG neurons to SP led to the inhibition of KCNQ K+ M-currents. The SP-mediated M-current block was significantly reduced in neurons transfected with Gα14 small-interference RNA. Finally, overexpression of the GTP-bound Gαq/11 binding protein RGS2 did not alter the block of M-currents by SP but significantly abolished the oxotremorine methiodide-mediated M-current inhibition. Taken together, these results provide evidence of a new Gα14-coupled signaling pathway that modulates CaV2.2 and M-currents via SP-stimulated NK-1 receptors in CSMG neurons. [ABSTRACT FROM AUTHOR]

Objective: Resveratrol, a phytoalexin, has a wide range of desirable biological actions. Despite a growing body of evidence indicating that resveratrol induces changes in neuronal function, little effort, if any, has been made to investigate the cellular effect of resveratrol treatment on intrinsic neuronal properties. Materials and Methods: This experimental study was performed to examine the acute effects of resveratrol (100 μM) on the intrinsic evoked responses of rat Cornu Ammonis (CA1) pyramidal neurons in brain slices, using whole cell patch clamp recording under current clamp conditions. Results: Findings showed that resveratrol treatment caused dramatic changes in evoked responses of pyramidal neurons. Its treatment induced a significant (P<0.05) increase in the after hyperpolarization amplitude of the first evoked action potential. Resveratrol-treated cells displayed a significantly broader action potential (AP) when compared with either control or vehicle-treated groups. In addition, the mean instantaneous firing frequency between the first two action potentials was significantly lower in resveratrol-treated neurons. It also caused a significant reduction in the time to maximum decay of AP. The rheobase current and the utilization time were both significantly greater following resveratrol treatment. Neurons exhibited a significantly depolarized voltage threshold when exposed to resveratrol. Conclusion: Results provide direct electrophysiological evidence for the inhibitory effects of resveratrol on pyramidal neurons, at least in part, by reducing the evoked neural activity. [ABSTRACT FROM AUTHOR]

Memory acquisition and synaptic plasticity are accompanied by changes in the intrinsic excitability of CA1 pyramidal neurons. These activity-dependent changes in excitability are mediated by modulation of intrinsic currents which alters the responsiveness of the cell to synaptic inputs. The afterhyperpolarization (AHP), a major contributor to the regulation of neuronal excitability, is reduced in animals that have acquired several types of hippocampus-dependent memory tasks and also following synaptic potentiation by high frequency stimulation. BK channels underlie the fast AHP and contribute to spike repolarization, and this AHP is reduced in animals that successfully acquired traceeyeblink conditioning. This suggests that BK channel function is activity-dependent, but the mechanisms are unknown. In this study, we found that blockade of BK channels with paxilline (10 µM) decreased IAHP amplitude and increased spike half-width and instantaneous frequency in response to a +100 pA depolarization. In addition, induction of long term potentiation (LTP) by theta burst stimulation (TBS) in CA1 pyramidal neurons reduced BK channel's contribution to IAHP, spike repolarization, and instantaneous frequency. This result indicates that BK channel activity is decreased following synaptic potentiation. Interestingly, blockade of mammalian target of rapamycin (MTORC1) with rapamycin (400 nM) following synaptic potentiation restored BK channel function, suggesting a role for protein translation in signaling events which decreased postsynaptic BK channel activity following synaptic potentiation. [ABSTRACT FROM AUTHOR]

The present study was aimed to explore correlation between the altered pain perception and Na + channel activity in diabetic animals as well as the effect of tetracaine on sensory neurons of diabetic rat. In streptozotocin-induced diabetic rats behavioral nociceptive parameters were assessed. The Na + current ( I Na ) was obtained using whole-cell voltage-clamp configuration in dorsal root ganglion (DRG) neurons isolated from diabetic rat ( in vitro ). In addition, the effects of tetracaine on altered Na + channel activity associated with diabetes in small DRG neurons were evaluated. After induction of diabetes mechanical allodynia, thermal hyperalgesia and Na + channel activity were altered significantly in 4th and 6th week in relation to the control. Altered pain parameters were in correlation with increased I Na in time-dependent manner. In comparison to age-matched control (−1.10 ± 0.20 nA) the I Na was found to be −2.49 ± 0.21 nA at 4th week and −3.71 ± 0.28 nA at 6th week. The increased activity of Na + channels was blocked by tetracaine even in diabetic condition. The depression of the I Na on tetracaine exposure was not sensitive to the voltage or time. The conductance curve shifted towards right around −8.0 mV. The alterations in neuropathic pain associated with diabetes and Na + channel activity has been clearly correlated in time-dependent manner. The I Na density was increased significantly with the progression of neuropathic pain. Local anesthetic, tetracaine potentially blocked the Na + channel activity in diabetic sensory neurons. [ABSTRACT FROM AUTHOR]

Introduction: Resveratrol possesses a wide range of biological effects. However, the cellular mechanisms of its effects are not fully understood. In the present study, the cellular actions of resveratrol on intrinsic electrophysiological properties of the rat hippocampal CA1 pyramidal neurons were examined. Methods: The spontaneous and evoked firing properties of CA1 pyramidal neurons in adult rats exposed to resveratrol (100 µM) were examined using whole cell patch clamp recording under current clamp condition and the results were compared with control and vehicle treated groups. Results: Treatment with resveratrol caused alterations in neuronal firing characteristics. Application of resveratrol shifted the resting membrane potential (RMP) towards hyperpolarizing voltage (from -58.62±0.89 mV in control to - 67.06±0.89 mV after resveratrol). The after hyperpolarization potential (AHP) amplitude was significantly increased following extracellular application of resveratrol (P < 0.001). It also induced a significant increase in the peak amplitude of action potential in response to 100-300 pA depolarizing current pulses (P<0.05). Furthermore, resveratrol-treated neurons displayed a significantly increased time to peak in response to 400 and 500 pA depolarizing currents, when compared with either control or vehicle-treated groups (P<0.05). In addition, rise time to half-amplitude as well as rise tau and decay tau of action potential were significantly increased following resveratrol application (P<0.01, P<0.01 and P<0.01, respectively). Conclusion: Treatment with resveratrol changes the action potential parameters, hyperpolarizes the RMP and reduces neuronal excitability and thereby may probably induce neuroprotective effects. [ABSTRACT FROM AUTHOR]

Using patch-clamp techniques, we studied the plasticity of acid-sensing ion channels (ASIC) and transient receptor potential V1 (TRPV1) channel function in dorsal root ganglia (DRG) neurons retrogradely labeled from the bladder. Saline (control) or cyclophosphamide (CYP) was given intraperitoneally on days 1, 3, and 5. On day 6, lumbosacral (LS, L6-S2) or thoracolumbar (TL, T13-L2) DRG were removed and dissociated. Bladders and bladder DRG neurons from CYP-treated rats showed signs of inflammation (greater myeloperoxidase activity; lower intramuscular wall pH) and increased size (whole cell capacitance), respectively, compared with controls. Most bladder neurons (>90%) responded to protons and capsaicin. Protons produced multiphasic currents with distinct kinetics, whereas capsaicin always triggered a sustained response. The TRPV1 receptor antagonist A-425619 abolished capsaicin-triggered currents and raised the threshold of heat-activated currents. Prolonged exposure to an acidic environment (pH range: 7.2 to 6.6) inhibited proton-evoked currents, potentiated the capsaicin-evoked current, and reduced the threshold of heat-activated currents in LS and TL bladder neurons. CYP treatment reduced density but not kinetics of all current components triggered by pH 5. In contrast, CYP-treatment was associated with an increased current density in response to capsaicin in LS and TL bladder neurons. Correspondingly, heat triggered current at a significantly lower temperature in bladder neurons from CYP-treated rats compared with controls. These results reveal that cystitis differentially affects TRPV1- and ASIC-mediated currents in both bladder sensory pathways. Acidification of the bladder wall during inflammation may contribute to changes in nociceptive transmission mediated through the TRPV1 receptor, suggesting a role for TRPV1 in hypersensitivity associated with cystitis. [ABSTRACT FROM AUTHOR]

Mammalian retina contains 15-20 different retinal ganglion cell (RGC) types, each of which is responsible for encoding different aspects of the visual scene. The encoding is defined by a combination of RGC synaptic inputs, the neurotransmitter systems used, and their intrinsic physiological properties. Each cell's intrinsic properties are defined by its morphology and membrane characteristics, including the complement and localization of the ion channels expressed. In this study, we examined the hypothesis that the intrinsic properties of individual RGC types are conserved among mammalian species. To do so, we measured the intrinsic properties of 16 morphologically defined rat RGC types and compared these data with cat RGC types. Our data demonstrate that in the rat different morphologically defined RGC types have distinct patterns of intrinsic properties. Variation in these properties across cell types was comparable to that found for cat RGC types. When presumed morphological homologs in rat and cat retina were compared directly, some RGC types had very similar properties. The rat A2 cell exhibited patterns of intrinsic properties nearly identical to the cat alpha cell. In contrast, rat D2 cells (ON-OFF directionally selective) had a very different pattern of intrinsic properties than the cat iota cell. Our data suggest that the intrinsic properties of RGCs with similar morphology and suspected visual function may be subject to variation due to the behavioral needs of the species. [ABSTRACT FROM AUTHOR]

Neuroinflammation has the capacity to alter normal central nervous system (CNS) homeostasis and function. The objective of the present study was to examine the effects of an inflammatory milieu on the electrophysiological properties of striatal astrocyte subpopulations with a mouse bacterial brain abscess model. Whole cell patch-clamp recordings were performed in striatal glial fibrillary acidic protein (GFAP)-green fluorescent protein (GFP)⁺ astrocytes neighboring abscesses at postinfection days 3 or 7 in adult mice. Cell input conductance (Gi) measurements spanning a membrane potential (Vm) surrounding resting membrane potential (RMP) revealed two prevalent astrocyte subsets. A1 and A2 astrocytes were identified by negative and positive Gi increments vs. Vm, respectively. A1 and A2 astrocytes displayed significantly different RMP, Gi, and cell membrane capacitance that were influenced by both time after bacterial exposure and astrocyte proximity to the inflammatory site. Specifically, the percentage of A1 astrocytes was decreased immediately surrounding the inflammatory lesion, whereas A2 cells were increased. These changes were particularly evident at postinfection day 7, revealing increased cell numbers with an outward current component. Furthermore, RMP was inversely modified in A1 and A2 astrocytes during neuroinflammation, and resting Gi was increased from 21 to 30 nS in the latter. In contrast, gap junction communication was significantly decreased in all astrocyte populations associated with inflamed tissues. Collectively, these findings demonstrate the heterogeneity of striatal astrocyte populations, which experience distinct electrophysiological modifications in response to CNS inflammation. [ABSTRACT FROM AUTHOR]

Abstract: Recurrent excitatory circuits and abnormal recurrent excitatory inputs are essential in epileptogenesis. Studies in temporal lobe epilepsy have shown that mossy fiber sprouting, which represents synaptic reorganization, renders the formation of abnormal recurrent excitatory circuits and inputs. The mammalian target of rapamycin (mTOR) pathway has recently been proved important in mossy fiber sprouting. In the present study, rapamycin, a mTOR inhibiter, was injected into the mouse of temporal lobe epilepsy. Electrophysiological and histological properties of the hippocampus were investigated by whole cell patch clamp, extracellular recording and Timm staining. Following the development of epilepsy, frequency of spontaneous excitatory postsynaptic currents (EPSCs) and amplitude of antidromically evoked EPSCs in granule cells were remarkably increased, as well as the epileptiform activity and mossy fiber sprouting were detected, which indicated the formation of abnormal recurrent excitatory circuits. By the use of rapamycin, frequency of spontaneous EPSCs, amplitude of antidromically evoked EPSCs, the epileptiform activity and mossy fiber sprouting were all remarkably suppressed. Our findings suggested an anti-epileptogenic role of rapamycin by suppressing the recurrent excitatory circuits of dentate gyrus. [Copyright &y& Elsevier]

In the nucleus accumbens (NAc), a medium spiny (MS) neuron receives GABAergic inputs from two major sources: fast-spiking (FS) neurons and other, adjacent MS neurons. These two types of inhibitory synapses are considered to play different roles in output activities, i.e., FS→MS connections suppress output from the NAc whereas MS→MS connections contribute to lateral inhibition. In the present study, we focused on the electrophysiological properties of unitary inhibitory postsynaptic currents (uIPSCs) obtained from MS→MS connections and FS→MS connections and examined the effects of quinpirole, a dopamine D2-like receptor agonist, on uIPSCs with multiple whole cell patch-clamp recording. Application of quinpirole (1 μM) reliably suppressed the amplitude of uIPSCs by 29.6% in MS→MS connections, with increases in pairedpulse ratio and failure rate. The suppressive effects of quinpirole on uIPSCs were mimicked by 1 μM PD128907, a D2/3 receptor agonist, whereas quinpirole-induced suppression of uISPCs was blocked by preapplication of 1 μM sulpiride or 10 μM nafadotride, both D2/3 receptor antagonists. On the other hand, quinpirole (1 μM) had divergent effects on FS→MS connections, i.e., quinpirole increased uIPSC amplitude in 38.1% of FS→MS connections and 23.8% of FS→MS connections were suppressed by quinpirole. Analysis of coefficient of variation in uIPSC amplitude implied the involvement of presynaptic mechanisms in quinpirole-induced effects on uIPSCs. These results suggest that activation of D2-like receptors facilitates outputs from MS neurons in the NAc by reducing lateral inhibition during a dormant period of FS neuron activities. [ABSTRACT FROM AUTHOR]

The development of central nervous system slice preparations for electrophysiological studies has led to an explosion of knowledge of neuronal properties in health and disease. Studies of spinal motoneurons in these preparations, however, have been largely limited to the early postnatal period, as adult motoneurons are vulnerable to the insults sustained by the preparation. We therefore sought to develop an adult spinal cord slice preparation that permits recording from lumbar motoneurons. To accomplish this, we empirically optimized the composition of solutions used during preparation in order to limit energy failure, reduce harmful ionic fluxes, mitigate oxidative stress, and prevent excitotoxic cell death. In addition to other additives, this involved the use of ethyl pyruvate, which serves as an effective nutrient and antioxidant. We also optimized and incorporated a host of previously published modifications used for other in vitro preparations, such as the use of polyethylene glycol. We provide an in-depth description of the preparation protocol and discuss the rationale underlying each modification. By using this protocol, we obtained stable whole cell patch-clamp recordings from identified fluorescent protein-labeled motoneurons in adult slices; here, we describe the firing properties of these adult motoneurons. We propose that this preparation will allow further studies of how motoneurons integrate activity to produce adult motor behaviors and how pathological processes such as amyotrophic lateral sclerosis affect these neurons. [ABSTRACT FROM AUTHOR]

Glucagon-like peptide-2 (GLP-2) is a neuropeptide secreted from endocrine cells in the gut and neurons in the brain. GLP-2 stimulates intestinal crypt cell proliferation and mucosal blood flow while decreasing gastric emptying and gut motility. However, a GLP-2 mediated signaling network has not been fully established in primary cells. Since the GLP-2 receptor mRNA and protein were highly expressed in the mouse hippocampus, we further characterized that human 125I-labeled GLP-21-33 specifically bound to cultured hippocampal neurons with Kd = 0.48 nM, and GLP-2 acutely induced subcellular translocalization of the early gene c-Fos. Using the whole cell patch clamp, we recorded barium currents (IBa) flowing through voltage-gated Ca2+ channels (VGCC) in those neurons in the presence of GLP-2 with and without inhibitors. We showed that GLP-2 (20 nM) enhanced the whole cell IBa mediated by L-type VGCC that was defined using an L-type Ca2+ channel blocker (nifedipine, 10 μM). Moreover, GLP-2-potentiation of L-type VGCC was abolished in neurons pretreated with a PKA inhibitor (PKI14-22, 1 μM). Finally, using a fluorescent nonmetabolized glucose analog (6-NBDG) tracing imaging, we showed that glucose was taken up directly by cultured neurons. GLP-2 increased 2-deoxy-D-[3H]glucose uptake that was dependent upon dosage, activation of PKA, and potentiation of L-type VGCC. We conclude that GLP-2 potentiates L-type VGCC activity through activating PKA signaling, partially stimulating glucose uptake by primary cultured hippocampal neurons. The potentiation of L-type VGCC may be physiologically relevant to GLP-2-induced neuroendocrine modulation of neurotransmitter release and hormone secretion. [ABSTRACT FROM AUTHOR]

Abstract: ASIC2a (BNaC1 or MDEG) is distributed throughout the nervous system and potentially involved in mechanosensation, hearing, vision, and taste functions. However, pharmacological properties of ASIC2 homomers including the mechanism of inhibition by amiloride remain unclear. In this study, we describe the properties of hASIC2a stably expressed in Ltk− cells, the first reported stable cell line expressing any ASICs subunit, by standard whole cell voltage clamp method. In response to pH 4.0, at −80 mV, hASIC2a cells exhibited rapidly activating fast transient inward current (∼100 pA/pF) that was followed by a sustained current (∼13 pA/pF). In contrast, untransfected Ltk− cells showed only a very small rapidly activating non-inactivating inward current (∼4 pA/pF). The magnitude of hASIC2a transient current was pH dependent with pH50 values for activation and inactivation of ∼4.2 and ∼5.5, respectively. Ion substitution experiments revealed the following rank order of permeability: Na+ >K+ >Ca2+ for the transient current. Amiloride reversibly inhibited the pH 4.0 evoked transient current with IC50 values of ∼20 μM at both −30 and −80 mV holding potentials, indicating that the interactions are voltage independent when nearly all amiloride is protonated. Amiloride (100 μM) did not inhibit ASIC2a transient current when pre-applied in pH 7.4 and pH 4.0 currents obtained in absence of amiloride, but it did inhibit currents when co-applied at pH 4.0 suggesting open channel blockade. In summary, ASIC2a stable cell line serves as a useful model system to study the pharmacological properties of ASIC2a currents, potentially contributing to pH-evoked responses in cells of the dorsal root ganglion and the central nervous system. [Copyright &y& Elsevier]

Abstract: A significant decline in executive system function mediated by the prefrontal cortex (PFC) often occurs with normal aging. In vitro slice studies have shown that layer 2/3 pyramidal cells in the monkey PFC exhibit increased action potential (AP) firing rates which may, in part, contribute to this decline. Given that layer 5 cells also play a role in executive system function, it is important to determine if similar age-related changes occur in these cells. Whole-cell patch-clamp recordings in in vitro slices prepared from the PFC of young and aged behaviorally characterized rhesus monkeys were employed to answer this question. Basic membrane and repetitive AP firing properties were unaltered with age. Aged cells exhibited significantly decreased single AP amplitude, duration and fall time and increased slow afterhyperpolarization (sAHP) amplitude, but these changes were not associated with cognitive performance. This study demonstrates that layer 5 pyramidal cells, unlike layer 2/3 pyramidal cells, undergo only modest electrophysiological changes with aging, and that these changes are unlikely to contribute to age-related cognitive decline. [Copyright &y& Elsevier]

Background: Data accumulated in the last decade indicate that N-methyl-D-aspartate (NMDA) receptors might be involved in the pathophysiology of depression and the mechanism of action of antidepressants, although a direct inhibitory effect has been reported only in connection with tricyclic compounds, which interact with a wide range of receptors. Methods: Using whole-cell patch-clamp recording in rat cortical cell cultures, we investigated whether the selective serotonin reuptake inhibitor fluoxetine, which has a much better adverse effect profile, has a direct effect on NMDA receptors, and we compared its action to that of the tricyclic desipramine. Results: Both desipramine (concentration that causes 50% inhibition (IC50) = 3.13 μM) and fluoxetine (IC50 = 10.51 μM) inhibited NMDA-evoked currents with similar efficacy in the clinically relevant low micromolar concentration range. However, in contrast to desipramine, the inhibition by fluoxetine was not voltage-dependent, and fluoxetine partially preserved its ability to associate with NMDA receptor in the presence of Mg2+, suggesting different binding sites for the two drugs. Conclusions: The fact that different classes of antidepressants were found to be low-affinity NMDA antagonists suggests that direct inhibition of NMDA receptors may contribute to the clinical effects of antidepressants. [Copyright &y& Elsevier]

Abstract: In the spinal dorsal horn, activation of the nicotinic acetylcholine receptors (nAChR) by exogenously applied agonists is known to enhance inhibitory synaptic transmission, and to produce analgesia. However, it is still unknown whether endogenously released acetylcholine exerts a tonic inhibition on nociceptive transmission through the nAChRs in the spinal dorsal horn. Here, we report the presence of such a tonic inhibitory mechanism in the spinal dorsal horn in mice. In behavioral experiments, intrathecal (i.t.) injection of non-selective nAChR antagonist mecamylamine and α4β2 subtype-selective antagonist dihydro-β-erythroidine (DHβE) dose-dependently induced thermal and mechanical hyperalgesia in mice while the α7-selective antagonist methyllycaconitine (MLA) had no effect. Similarly, antisense knock-down of α4 subunit of nAChR, but not α7 subunit, in spinal cord induced thermal and mechanical hyperalgesia. In whole-cell patch-clamp experiments in spinal cord slice preparation from adult mice, the frequency of miniature inhibitory postsynaptic currents (mIPSCs) observed in substantia gelatinosa (SG) neurons was decreased by mecamylamine and DHβE, but not by MLA. The amplitudes of the mIPSCs were not affected. The nicotinic antagonists decreased the frequency of both GABAergic and glycinergic IPSCs. On the other hand, the nicotinic antagonists had no effect on the excitatory postsynaptic currents (EPSCs). Finally, acetylcholine-esterase inhibitor neostigmine-induced facilitation of IPSC frequencies in SG neurons was inhibited by mecamylamine and DHβE. Altogether these findings suggest that nicotinic cholinergic system in the spinal dorsal horn can tonically inhibit nociceptive transmission through presynaptic facilitation of inhibitory neurotransmission in SG via the α4β2 subtype of nAChR. [Copyright &y& Elsevier]

Abstract: Previous studies on dorsal motor nucleus of the vagus (DMNV) neurons have mainly used in vivo animal models and in vitro brainstem slices. Primary culture of postnatal DMNV neurons in defined serum free medium has not been reported. We report a method for culture of postnatal rat DMNV neurons using serum free medium. Cultured DMNV neurons contain both Hu positive precursor cells and mature cells staining positively for microtubule associated protein 2 (MAP2) and choline acetyltransferase. Exposure of cultured DMNV neurons to glutamate (10−7 to 10−3 M) induced an increase in intracellular calcium concentration ([Ca2+] i ) in a dose-dependent manner, indicating the functional presence of glutamate receptors. Voltage-dependent calcium currents were present in cultured DMNV neurons. Active cell proliferation was demonstrated by BrdU incorporation. Upon removal of βFGF, the percentage of MAP2 positive mature neurons was significantly increased from 36±3 to 73±3%. Our study demonstrates that postnatal rat DMNV neurons cultured in serum free medium retain morphological and physiological characteristics of DMNV neurons in situ. [Copyright &y& Elsevier]

Abstract: Pregnenolone sulfate is one of the most abundantly produced neurosteroids in the brain. The present paper studies the effect of pregnenolone sulfate on excitatory synaptic transmission in the pyramidal cells of the layer V–VI of the prelimbic cortex using whole-cell patch-clamp in slices. We found that pregnenolone sulfate inhibited stimulus-evoked excitatory postsynaptic currents (EPSC); the effect of pregnenolone sulfate was significant at concentration of 1 μM and increased with an increase in concentrations; pregnenolone sulfate had no effect on the amplitude and frequency of miniature excitatory spontaneous postsynaptic currents; pregnenolone sulfate significantly enhanced the paired-pulse facilitation (PPF) and inhibited dopamine and 5-HT-evoked increase in the frequency of spontaneous excitatory postsynaptic currents; the protein kinase A inhibitor H89 and Rp-cAMPS enhanced PPF and canceled the effect of pregnenolone sulfate on PPF; pregnenolone sulfate inhibited the protein kinase A agonist forskolin-evoked increase in the frequency of spontaneous excitatory postsynaptic currents; the Gi protein inhibitor N-ethylmaleimide canceled the effect of pregnenolone sulfate on PPF. These results suggest that pregnenolone sulfate inhibits stimulus-evoked EPSC via presynaptic inhibition of protein kinase A in rat prelimbic cortical neurons. [Copyright &y& Elsevier]

Inhibition at bipolar cell axon terminals regulates excitatory signaling to ganglion cells and is mediated, in part, by GABAC receptors. We investigated GABAC receptor-mediated inhibition using pharmacological approaches and genetically altered mice that lack GABAC receptors. Responses to applied GABA showed distinct time courses in various bipolar cell classes, attributable to different proportions of GABAA and GABAC receptors. The elimination of GABAC receptors in GABAC null mice reduced and shortened GABA-activated currents and light-evoked inhibitory synaptic currents (L-IPSCs) in rod bipolar cells. ERG measurements and recordings from the optic nerve showed that inner retinal function was altered in GABAC null mice. These data suggest that GABAC receptors determine the time course and extent of inhibition at bipolar cell terminals that, in turn, modulates the magnitude of excitatory transmission from bipolar cells to ganglion cells. [Copyright &y& Elsevier]

In excitable cells, hypoxia inhibits K channels, causes membrane depolarization, and initiates complex adaptive mechanisms. It is unclear whether K channels of alveolar epithelial cells reveal a similar response to hypoxia. A549 cells were exposed to hypoxia during whole cell patch-clamp measurements. Hypoxia reversibly inhibited a voltage-dependent outward current, consistent with a K current, because tetraethylamonium (TEA; 10 mM) abolished this effect; however, iberiotoxin (0.1 μM) does not. In normoxia, TEA and iberiotoxin inhibited whole cell current (-35%), whereas the K-channel inhibitors glibenclamide (1 μM), barium (1 mM), chromanol B293 (10 μM), and 4-aminopyridine (1 μM) were ineffective. 86Rb uptake was measured to see whether K-channel modulation also affected transport activity. TEA, iberiotoxin, and 4-h hypoxia (1.5% O2) inhibited total 86Rb uptake by 40, 20, and 35%, respectively. Increased extracellular K also inhibited 86Rb uptake in a dose-dependent way. The K-channel opener 1-ethyl-2-benzimidazolinone (1 mM) increased 86Rb uptake by 120% in normoxic and hypoxic cells by activation of Na-K pumps (+60%) and Na-K-2CI cotransport (+170%). However, hypoxic transport inhibition was also seen in the presence of l-ethyl-2-benzimidazolinone, TEA, and iberiotoxin. These results indicate that hypoxia, membrane depolarization, and K-channel inhibition decrease whole cell membrane currents and transport activity. It appears, therefore, that a hypoxia-induced change in membrane conductance and membrane potential might be a link between hypoxia and alveolar ion transport inhibition. [ABSTRACT FROM AUTHOR]

In the peripheral nervous system, N-methyl-d-aspartate receptors (NMDAR) expressed on the central and peripheral terminals of primary afferent neurons are involved in nociception. We used single cell imaging of intracellular calcium concentration ([Ca2+]i) and patch clamp techniques to characterize the functional properties of NMDARs on adult rat dorsal root ganglia (DRG) neurons in primary culture and selectively on those innervating the distal colon. In Mg2+-free extracellular solution, rapid perfusion of DRG neurons with 250 μM NMDA and 10 μM glycine caused a significant increase in [Ca2+]i, and elicited inward currents in whole cell patch clamp recordings when the holding potential was -60 mV. Both effects were reversibly inhibited by 200 μM ketamine in a use-dependent manner. The EC50 values for NMDA and glycine were 64 and 1.9 μM with Hill slope coefficients of 1.4 and 1.3, respectively. At negative potentials, extracellular Mg2+ blocked currents in a concentration- and voltage-dependent manner. The IC50 for Mg2+ at a holding potential of -100 mV was 2.0 μM. The NMDAR subtype-selective antagonist, ifenprodil, inhibited 94% of the NMDA and glycine-induced current with an IC50 of 2.6 μM. There was no evidence of multiple binding sites for ifenprodil. There was no significant difference in the NMDAR current density on DRG neurons that had innervated the colon, nor was there a difference in the EC50 for ifenprodil. These results demonstrate that functional NMDARs expressed by DRG neurons innervating both somatic and visceral tissues of adult rats are composed predominantly of NR2B subunits. [Copyright &y& Elsevier]

The two major inputs to CA1 pyramidal neurons, the perforant pathway (PP) that terminates on distal dendrites and the Schaffer collaterals (SCH) that terminate on proximal dendrites, activate both AMPA and N-methyl-D-aspartate (NMDA) receptors. In an in vitro slice preparation, the pharmacologically isolated NMDA receptor-mediated excitatory postsynaptic currents (EPSCs) (NMDA-EPSCs) of either pathway can be selectively activated onto a single CA1 pyramidal neuron. Analysis of the decay phase of PP and SCH NMDA-EPSCs revealed no significant difference in their time constants, suggesting no apparent different distribution in NR2-subunit composition in the NMDA receptors (NMDAR) activated by the two synaptic inputs. However, application of the NR2B-selective antagonist, ifenprodil, differently affected the NMDA-EPSCs activated by the PP and SCH inputs. The reduction of the PP responses was only 30% compared to 75% for the SCH responses. In addition, for both pathways, the ifenprodil-insensitive component of the NMDA-EPSCs had significantly more rapid decay kinetics than those prior to application of ifenprodil. Our results show a greater NR2B subunit contribution to the NMDA component of the SCH EPSC, compared to the NMDA component of the PP EPSC and that in single CA1 pyramidal neurons NMDA composition is anatomically specific to the afferent input. [ABSTRACT FROM AUTHOR]

Uremic retention solutes possibly contribute to neuronal hypoxia/ischemia and its consequences in patients with renal failure. We examined the in vitro effects of several uremic retention solutes on murine central neurons under chemically induced metabolic hypoxia by application of sodium cyanide (NaCN). Whole cell currents were recorded using the tight-seal whole-cell voltage clamp technique. Application of NaCN caused an inward whole-cell current. From all tested toxins, which included several indoles, guanidino compounds, polyamines, purines, phenols, dl-homocysteine, orotate and myoinositol, only creatinine (CTN), guanidine (G) and guanidinosuccinic acid (GSA) produced a significant current in control and hypoxic neurons. Current evoked by GSA was significantly increased in the chemical hypoxic condition, and a synergistic effect of GSA and spermine was observed in hypoxic neurons. [Copyright &y& Elsevier]

Electrophysiological studies have revealed an increase in the level of tonic inhibition in the hippocampus following prenatal protein malnutrition in rats. In the present study, whole cell patch clamp recordings of bipolar interneurons in the stratum radiatum of the CA3 subfield were used to determine whether this increase in inhibition can be accounted for by a change in the electrophysiological properties of GABAergic interneurons. Hippocampal slices were prepared from juvenile rats whose dams were fed either a normal (25% casein) or low (6% casein) protein diet throughout pregnancy. Intrinsic membrane and action potential properties were unaltered by the prenatal nutritional insult. In most respects the characteristics of GABA A receptor-mediated inhibitory postsynaptic currents (IPSCs) and the modulation of such currents by the benzodiazepine agonist zolpidem were also similar in cells from the two nutritional groups. While the frequency of spontaneous inhibitory currents was unaltered, miniature (Tetrodotoxin resistant) inhibitory currents occurred at a significantly increased frequency in interneurons from prenatally protein malnourished rats. Thus, while the basic membrane properties of interneurons are preserved, there is a significant increase in the probability of GABA release from interneurons following prenatal protein malnutrition. [ABSTRACT FROM AUTHOR]

We studied chemosensitive signaling in locus coeruleus (LC) neurons using both perforated and whole cell patch techniques. Upon inhibition of fast Na+ spikes by tetrodotoxin (TTX), hypercapnic acidosis [HA; 15% CO2, extracellular pH (pHo) 6.8] induced small, slow spikes. These spikes were inhibited by Co2+ or nifedipine and were attributed to activation of L-type Ca2+ channels by HA. Upon inhibition of both Na+ and Ca2+ spikes, HA resulted in a membrane depolarization of 3.52 ± 0.61 mV (n = 17) that was reduced by tetraethylammonium (TEA) (1.49 ± 0.70 mV,n = 7; P < 0.05) and absent (−0.97 ± 0.73 mV, n = 7; P < 0.001) upon exposure to isohydric hypercapnia (IH; 15% CO2, 77 mM HCO−3, pHo 7.45). Either HA or IH, but not 50 mM Na-propionate, activated Ca2+ channels. Inhibition of L-type Ca2+ channels by nifedipine reduced HA-induced increased firing rate and eliminated IH-induced increased firing rate. We conclude that chemosensitive signals (e.g., HA or IH) have multiple targets in LC neurons, including TEA-sensitive K+ channels and TWIK-related acid-sensitive K+ (TASK) channels. Furthermore, HA and IH activate L-type Ca2+ channels, and this activation is part of chemosensitive signaling in LC neurons. [ABSTRACT FROM AUTHOR]

Epithelial cells from the anterior and equatorial surfaces of the frog lens were isolated and used the same day for studies of the Na/K ATPase. RNase protection assays showed that all cells express alpha(1)- and alpha(2)-isoforms of the Na/K pump but not the alpha(3)-isoform, however the alpha(2)-isoform dominates in anterior cells whereas the alpha(1)-isoform dominates in equatorial cells. The whole cell patch-clamp technique was used to record functional properties of the Na/K pump current (I(P)), defined as the current specifically inhibited by dihydro-ouabain (DHO). DHO-I(P) blockade data indicate the alpha(1)-isoform has a dissociation constant of 100 microm DHO whereas for the alpha(2)-isoform it is 0.75 microm DHO. Both alpha(1)- and alpha(2)-isoforms are half maximally activated at an intracellular Na(+)-concentration of 9 mm. The alpha(1)-isoform is half maximally activated at an extracellular K(+)-concentration of 3.9 mm whereas for the alpha(2)-isoform, half maximal activation occurs at 0.4 mm. Lastly, transport by the alpha(1)-isoform is inhibited by a drop in extracellular pH, which does not affect transport by the alpha(2)-isoform. Under normal physiological conditions, I(P) in equatorial cells is approximately 0.23 microA/microF, and in anterior cells it is about 0.14 microA/microF. These current densities refer to the area of cell membrane assuming a capacitance of around 1 microF/cm(2). Because cell size and geometry are different at the equatorial vs. anterior surface of the intact lens, we estimate Na/K pump current density per area of lens surface to be around 10 microA/cm(2) at the equator vs. 0.5 microA/cm(2) at the anterior pole. [ABSTRACT FROM AUTHOR]

Objective: To investigate the electrophysiological properties of pyramidal neurons in mouse motor cortex during the early postnatal development., Methods: Thirty-six mice were randomly divided into postnatal 1-, 2-, 3-Week and 1-, 2-,3-Month groups ( n =6). Membrane properties, action potentials (AP) and spontaneous excitatory postsynaptic currents (sEPSCs) of motor cortex pyramidal neurons were recorded to evaluate the changes in the intrinsic electrophysilogical characteristics by using whole cell patch clamp. Pyramidal neurons and interneurons were distinguished according to the AP firing patterns., Results: Comparing with interneurons, pyramidal neurons exhibited regular spiking (RS) with smaller frequency. During the period of postnatal 1 Week-3 Months, some of the intrinsic membrane properties of motor cortex pyramidal neurons changed. Compared to the 1-Week mice, the resting membrane potential (RMP) of 2-Week decreased significantly ( P ＜0.01), and the membrane input resistance (R in ) of 1-Month got a hyperpolarization ( P ＜0.01), and they showed no significant change in the next period, while the membrane capacitance (Cm) showed no significant changes during the whole postnatal development. The AP dynamic properties changed significantly during this period. Compared to the 1-Week mice, the absolute value of the AP threshold and the AP amplitude of the 3-Week increased significantly ( P ＜0.01), while the spike half width of the 2-Week decreased substantially ( P ＜0.05), and they showed no significant change in the next period. The sEPSCs frequency and amplitude of 1- Month increased significantly compared to the 1-Week mice( P ＜0.01), while during the period of next 1 Month-3 Months, the amplitude and frequency showed no significant change., Conclusion: These results suggest that the motor cortex pyramidal neurons have time-specific eletrophysilogical properties during the postnatal development. The electrophysiological properties can be used as a functional index to detect the degree of neurons maturity, and as a marker to distinguish the pyramidal neurons and interneurons.