Decrease In Membrane Conductance Research Articles

ДЕЙСТВИЕ ЭКСТРАКТОВ ИЗ ARTEMİSİA L. НА ПЛАЗМАТИЧЕСКУЮ МЕМБРАНУ В УСЛОВИЯХ СТРЕССА

Plant bioactive compounds are supposed to be the modifiers of structural-functional properties of cell plasma membrane under HM contamination. To test a protective effect of extracts from Artemisia L. species, possessing high adaptive potential to different soil contaminations, on electrical properties of algae cells under HM treatments was the aim of the present work. Extracts from Artemisia plants were isolated from above-ground parts of A.scoparia and A.szovitsiana by acetone hydrolyzation. Microelectrode techniques were used to study the regularities of change of membrane potential and membrane resistance of Chara gymnophylla and Nitella flexilis plasmalemma under influence of Cd, Cu, Zn, Pb and the extracts from Artemisia species. Statistically reliable hyperpolarizing effect of A.szovitsiana and A.scoparia extracts in relation to Zn and Pb was revealed, respectively. Elimination by extracts a decrease of membrane conductivity caused by Cd and Zn and also an increase of conductivity by Cu was determined. Though a high HM accumulation capacity of Artemisia L. species is noticed to not connect with protective effect of their extracts on membrane conductivity, they can be a potential source for the searching new chemical compounds which are capable to induce the plant tolerance to stress impacts.

Cannabidiol counters the effects of a dominant-negative pathogenic Kv7.2 variant

SummaryEpilepsy and neurodevelopmental disorders can arise from pathogenic variants of KCNQ (Kv7) channels. A patient with developmental and epileptic encephalopathy exhibited an in-frame deletion of histidine 260 on Kv7.2. Coexpression of Kv7.2 mutant (mut) subunits with Kv7.3 invoked a decrease in current density, a depolarizing shift in voltage for activation, and a decrease in membrane conductance. Biotinylation revealed an increased level of surface Kv7.2mut compared to Kv7.3 with no change in total membrane protein expression. Super-resolution and FRET imaging confirmed heteromeric channel formation and a higher expression density of Kv7.2mut. Cannabidiol (1 μM) offset the effects of Kv7.2mut by inducing a hyperpolarizing shift in voltage for activation independent of CB1 or CB2 receptors. These data reveal that the ability for cannabidiol to reduce the effects of a pathogenic Kv7.2 variant supports its use as a potential therapeutic to reduce seizure activity.

Noninvasive Measurement of Conductivity Distribution in Polymer Electrolyte Fuel Cells (PEFCs)

Water management in Polymer Electrolyte Fuel Cells (PEFCs) is a crucial topic. Indeed, a homogeneous distribution of the water in the membrane plane is important as it dictates its conductivity. Furthermore, local dry outs or flooding event can damage the cell. Existing measurement methods that are able to provide spatially resolved information on the current density, conductivity or water content distribution are usually invasive and therefore difficult to use in actual applications. A new method, based on the learnings of the development of Electrical Impedance Tomography (EIT) for PEFCs [Schuller and Eller 2020 ECS Trans. 98 3], is used to determine 1D humidity profiles between the gas inlet and outlet in a 6-cell commercial stack. Local AC current injections and voltage measurements are performed using electrodes positioned all around the fuel cell. Each measurement characterizes a defined area of the membrane as the current crosses it very locally. In a first step, the method is calibrated using nitrogen flow at different well-defined humidity levels. Using the calibration data the local boundary voltages can be converted into local humidity conditions when the stack is operated. In Figure 1a the reconstructed conductivity distributions of the stack being operated at four different current densities with inlet relative humidity (RH) of 60 % at the anode and 30 % at the cathode in counter flow configuration is shown. The y-axis represents the relative membrane conductivity value where 1 corresponds to the reference measurement with the highest inlet RH (90%). The conductivity increases with increasing current densities and there tends to be a higher conductivity towards the cathode outlet due to water drag through the membrane. Thanks to the fast multi-channel data acquisition setup, the method can be also used to study the dynamic behavior of a cell. Figure 1b shows the time resolved reconstruction of the conductivity distribution when flushing the cell with nitrogen (anode and cathode at 40% RH) after the stack is operated at 0.8 A/cm2. The method is capable to resolve the quick decrease in membrane conductivity at both inlets and to follow the much slower drying process of the membrane in the center of the cell. Figure 1

Dielectrophoresis as a Tool to Reveal the Potential Role of Ion Channels and Early Electrophysiological Changes in Osteoarthritis.

Diseases such as osteoarthritis (OA) are commonly characterized at the molecular scale by gene expression and subsequent protein production; likewise, the effects of pharmaceutical interventions are typically characterized by the effects of molecular interactions. However, these phenomena are usually preceded by numerous precursor steps, many of which involve significant ion influx or efflux. As a consequence, rapid assessment of cell electrophysiology could play a significant role in unravelling the mechanisms underlying drug interactions and progression of diseases, such as OA. In this study, we used dielectrophoresis (DEP), a technique that allows rapid, label-free determination of the dielectric parameters to assess the role of potassium ions on the dielectric characteristics of chondrocytes, and to investigate the electrophysiological differences between healthy chondrocytes and those from an in vitro arthritic disease model. Our results showed that DEP was able to detect a significant decrease in membrane conductance (6191 ± 738 vs. 8571 ± 1010 S/m2), membrane capacitance (10.3 ± 1.47 vs. 14.5 ± 0.01 mF/m2), and whole cell capacitance (5.4 ± 0.7 vs. 7.5 ± 0.3 pF) following inhibition of potassium channels using 10 mM tetraethyl ammonium, compared to untreated healthy chondrocytes. Moreover, cells from the OA model had a different response to DEP force in comparison to healthy cells; this was seen in terms of both a decreased membrane conductivity (782 S/m2 vs. 1139 S/m2) and a higher whole cell capacitance (9.58 ± 3.4 vs. 3.7 ± 1.3 pF). The results show that DEP offers a high throughput method, capable of detecting changes in membrane electrophysiological properties and differences between disease states.

Acute effects of hypertonic saline inhalation on nitric oxide pulmonary diffusing capacity in healthy adults

We investigated acute effects of inhalation of hypertonic saline solution (HSS) and oxygen (O2, control exposure) on pulmonary diffusing capacity for nitric oxide (DLNO) and carbon monoxide (DLCO). In a randomized crossover study, 20 healthy, non-smoking subjects were allocated to short-term inhalation of HSS or O2. Spirometry [(forced expiratory volume in 1 s (FEV1) and forced vital capacity (FVC)] and combined single-breath DLNO-DLCO measurements were performed before and immediately after inhalation of either HSS or O2. Percent changes were presented as median values (interquartile range). After HSS inhalation, DLNO, FEV1 and FVC were decreased by −3.0% (−7.3, 0.5), −3.1% (−4.2, −1.6) and −1.2% (−3.3, 0.6), respectively (all P < 0.05), without significant effect on DLCO. No changes in spirometry and diffusing capacity were observed following O2 inhalation. Acute inhalation of HSS causes a slight decrease in membrane conductance, probably as a result of fluid imbalance at the alveolar surface and interstitial fluid accumulation, both of which could impair gas exchange.

Nicotine inhibits potassium currents in Aplysia bag cell neurons.

Acetylcholine and the archetypal cholinergic agonist, nicotine, are typically associated with the opening of ionotropic receptors. In the bag cell neurons, which govern the reproductive behavior of the marine snail, Aplysia californica, there are two cholinergic responses: a relatively large acetylcholine-induced current and a relatively small nicotine-induced current. Both currents are readily apparent at resting membrane potential and result from the opening of distinct ionotropic receptors. We now report a separate current response elicited by applying nicotine to cultured bag cell neurons under whole cell voltage-clamp. This current was ostensibly inward, best resolved at depolarized voltages, presented a noncooperative dose-response with a half-maximal concentration near 1.5 mM, and associated with a decrease in membrane conductance. The unique nicotine-evoked response was not altered by intracellular perfusion with the G protein blocker GDPβS or exposure to classical nicotinic antagonists but was occluded by replacing intracellular K(+) with Cs(+) Consistent with an underlying mechanism of direct inhibition of one or more K(+) channels, nicotine was found to rapidly reduce the fast-inactivating A-type K(+) current as well as both components of the delayed-rectifier K(+) current. Finally, nicotine increased bag cell neuron excitability, which manifested as reduction in spike threshold, greater action potential height and width, and markedly more spiking to continuous depolarizing current injection. In contrast to conventional transient activation of nicotinic ionotropic receptors, block of K(+) channels could represent a nonstandard means for nicotine to profoundly alter the electrical properties of neurons over prolonged periods of time.

State-of-health of a CuCl electrolyzer during a 168-h test

A state-of-health test of a CuCl/HCl electrolytic cell was carried out by maintaining a constant current density of 0.3 A cm−2 for 168 h at 80 °C and ambient pressure. While the most common method used to characterize the electrochemical performance of a CuCl electrolytic cell is to record current–voltage (I–V curves), it is also essential to separate the electrochemical and ohmic components of the polarization curve. In our study, the AC impedance characteristics of a single cell were studied along with the DC polarization curves. After the durability test, the potential required to maintain the cell at 0.3 A cm−2 increased from 630 mV of the fresh membrane electrode assembly (MEA) to 710 mV due to some degradation processes in the electrolytic cell. Quantitative analysis of the performance decay was carried out by Electrochemical Impedance Spectroscopy (EIS). EIS measurements showed an increase in the area specific ohmic resistance of the MEA from 0.6 Ω cm2 to 0.85 Ω cm2 and area specific charge-transfer resistance from 0.61 Ω cm2 to 0.8 Ω cm2 measured at an overpotential of 0.2 V. The degradation in the cell is attributed to the decrease in membrane conductivity and an increase in the charge-transfer resistance.

Blockade of voltage-gated K+ currents in rat mesenteric arterial smooth muscle cells by MK801

MK801 (dizocilpine), a phencyclidine (PCP) derivative, is a potent noncompetitive antagonist of the N-Methyl-D-aspartate receptor (NMDAr). Another PCP derivative, ketamine, was reported to block voltage-gated K+ (Kv) channels, which was independent of NMDAr function. Kv currents are major regulators of the membrane potential (Em) and excitability of muscles and neurons. Here, we investigated the effect of MK801 on the Kv channels and Em in rat mesenteric arterial smooth muscle cells (RMASMCs). We used the whole-cell patch clamp technique to analyze the effect of MK801 enantiomers on Kv channels and Em. (+)MK801 inhibited Kv channels in a concentration-dependent manner (IC50 of 89.1 ± 13.1 μM, Hill coefficient of 1.05 ± 0.08). The inhibition was voltage- and state- independent. (+)MK801 didn't influence steady-state activation and inactivation of Kv channels. (+)MK801 treatment depolarized Em in a concentration-dependent manner and concomitantly decreased membrane conductance. (−)MK801 also similarly inhibited the Kv channels (IC50 of 134.0 ± 17.5 μM, Hill coefficient of 0.87 ± 0.09). These results indicate that MK801 directly inhibits the Kv channel in a state-independent manner in RMASMCs. This MK801-mediated inhibition of Kv channels should be considered when assessing the various pharmacological effects produced by MK801, such as schizophrenia, neuroprotection, and hypertension.

Impact of Structural Plastics as Balance of Plant Components on Proton Exchange Membrane Fuel Cell Performance

The impact of plastic material (Polyamide 610) as a balance of plant component on PEMFC (Proton Exchange Membrane Fuel Cell) performance was investigated in this paper. It is shown that this plastic material leaches out anions (chloride and nitrate) and cations (silicon and sodium) and low molecular weight compounds (polyamide 610 cyclic dimer and caprolactam) after 6 weeks of soaking time. The leachants result in significant loss of available Pt sites and decrease of membrane conductivity. The infusion test results show that the cell voltage loss decreases linearly with increased Pt loading while it increases linearly with increased leachant concentration. Increase of temperature also increases cell voltage loss. Although, the cell performance successfully recovers ~61%, the loss still does not meet strict automotive durability targets. Various types of plastic materials are being screened in order to minimize the adverse impact on fuel cell performance.

GIRK-like and TRPC-like conductances mediate thyrotropin-releasing hormone-induced increases in excitability in thalamic paraventricular nucleus neurons

The thalamic paraventricular nucleus (PVT), reported to participate in arousal and motivated behaviors, contains abundant receptors for thyrotropin-releasing hormone (TRH), a neuropeptide also known to modulate arousal and mood. To test the hypothesis that TRH could influence the excitability of PVT neurons, whole cell patch-clamp recordings obtained in rat brain slice preparations were evaluated during bath applied TRH. In the majority of neurons tested, TRH induced reversible TTX-resistant membrane depolarization. Under voltage-clamp, TRH induced a concentration-dependent G protein- mediated inward current. The mean net TRH-induced current exhibited a decrease in membrane conductance. Further analyses identified two concurrent conductances contributing to the TRH-induced response. One conductance featured a Na+-independent and K+-dependent net current that displayed rectification and was suppressed by micromolar concentrations of Ba2+ and two GIRK antagonists, tertiapin Q and SCH 23390. The second conductance featured a Na+-dependent net inward current with an I–V relationship that exhibited double rectification with a negative slope conductance below −40 mV. This conductance was suppressed by nonselective TRPC channel blockers 2-APB, flufenamic acid and ML204, enhanced by La3+ in a subpopulation of cells, and unchanged by the TRPV1 antagonist capsazepine or a Na+/Ca2+ exchanger blocker KB-R7943. TRH also enhanced hyperpolarization-activated low threshold spikes, a feature that was sensitive to pretreatment with either 2-APB or ML204. Collectively, the data imply that TRH enhances excitability in PVT neurons via concurrently decreasing a G-protein-gated inwardly rectifying K+ conductance and activating a cationic conductance with characteristics reminiscent of TRPC-like channels, possibly involving TRPC4/C5 subunits.

Abstract 18448: Partial Blockade of Inwardly Rectifying K + Current Enhances Electrotonic Interactions and Destabilizes Spiral Wave Reentry

Introduction: Spiral wave (SW) reentry is a principal mechanism for the maintenance of tachyarrhythmias, such as ventricular tachycardia/fibrillation (VT/VF). We have recently reported that partial blockade of the inward rectifier K + current (I K1 ) destabilizes the rotation center of SWs without enhancement of wavefront-tail interaction in the arm, in favor of their termination (ESC 2012), but underling mechanisms remain to be clarified. We hypothesized that alteration in electrotonic influence due to decreased membrane conductance plays a role in destabilization of the SW rotation center. Methods: A 2-dimensional ventricular muscle layer was prepared in Langendorff-perfused rabbit hearts, and optical action potential signals were recorded from the epicardial surface of the left ventricle. The dynamics of SW reentry were analyzed during VT induced by modified cross-field stimulation. The space constant ( λ ) was estimated by a decay of electrotonic depolarization induced by injection of subthreshold cathodal current. Results: Partial blockade of I K1 by 50 µM Ba 2+ significantly increased theλ: e.g., the value in a direction across the fiber orientation (λ T ) increased from 0.82±0.19 mm in control to 1.27±0.33 mm after 50 µM Ba 2+ (n=5, P&lt;0.05, Figure A). SW reentry under control condition rotated around a short line of block (FBL, 4.2±1.4 mm, n=12). In contrast, SW reentry induced after 50 µM Ba 2+ was destabilized: the FBL was significantly prolonged (11.7±3.2 mm, n=12, P&lt;0.05) and, in 6/12 hearts, showed a prominent change in its shape with a cycle of a few rotations, resulting from intermittent conduction block near the rotation center (Figure B). Conclusions: These results suggest that partial I K1 blockade enhances electrotonic interactions, which could contribute to destabilization of SW reentry.

CaMKII inhibition hyperpolarizes membrane and blocks nitrergic IJP by closing a Cl−conductance in intestinal smooth muscle

The ionic basis of nitrergic "slow'" inhibitory junction potential (sIJP) is not fully understood. The purpose of the present study was to determine the nature and the role of calmodulin-dependent protein kinase II (CaMKII)-dependent ion conductance in nitrergic neurotransmission at the intestinal smooth muscle neuromuscular junction. Studies were performed in guinea pig ileum. The modified Tomita bath technique was used to induce passive hyperpolarizing electrotonic potentials (ETP) and membrane potential change due to sIJP or drug treatment in the same cell. Changes in membrane potential and ETP were recorded in the same smooth muscle cell, using sharp microelectrode. Nitrergic IJP was elicited by electrical field stimulation in nonadrenergic, noncholinergic conditions and chemical block of purinergic IJP. Modification of ETP during hyperpolarization reflected active conductance change in the smooth muscle. Nitrergic IJP was associated with decreased membrane conductance. The CAMKII inhibitor KN93 but not KN92, the Cl(-) channel blocker niflumic acid (NFA), and the K(ATP)-channel opener cromakalim hyperpolarized the membrane. However, KN93 and NFA were associated with decreased and cromakalim was associated with increased membrane conductance. After maximal NFA-induced hyperpolarization, hyperpolarization associated with KN93 or sIJP was not seen, suggesting a saturation block of the Cl(-) channel signaling. These studies suggest that inhibition of CaMKII-dependent Cl(-) conductance mediates nitrergic sIJP by causing maximal closure of the Cl(-) conductance.

Neurotensin inhibits background K+ channels and facilitates glutamatergic transmission in rat spinal cord dorsal horn

Neurotensin (NT) is a neuropeptide involved in the modulation of nociception. We have investigated the actions of NT on cultured postnatal rat spinal cord dorsal horn (DH) neurons. NT induced an inward current associated with a decrease in membrane conductance in 46% of the neurons and increased the frequency of glutamatergic miniature excitatory synaptic currents in 37% of the neurons. Similar effects were observed in acute slices. Both effects of NT were reproduced by the selective NTS1 agonist JMV449 and blocked by the NTS1 antagonist SR48692 and the NTS1/NTS2 antagonist SR142948A. The NTS2 agonist levocabastine had no effect. The actions of NT persisted after inactivation of G(i/o) proteins by pertussis toxin but were absent after inactivation of protein kinase C (PKC) by chelerythrine or inhibition of the MAPK (ERK1/2) pathway by PD98059. Pre- and postsynaptic effects of NT were insensitive to classical voltage- and Ca(2+) -dependent K(+) channel blockers. The K(+) conductance inhibited by NT was blocked by Ba(2+) and displayed no or little inward rectification, despite the presence of strongly rectifying Ba(2+) -sensitive K(+) conductance in these neurons. This suggested that NT blocked two-pore domain (K2P) background K(+) -channels rather than inwardly rectifying K(+) channels. Zn(2+) ions, which inhibit TRESK and TASK-3 K2P channels, decreased NT-induced current. Our results indicate that in DH neurons NT activates NTS1 receptors which, via the PKC-dependent activation of the MAPK (ERK1/2) pathway, depolarize the postsynaptic neuron and increase the synaptic release of glutamate. These actions of NT might modulate the transfer and the integration of somatosensory information in the DH.

Two mechanisms underlie the slow noradrenergic depolarization in the rat tail artery in vitro

In rat tail artery, short trains of electrical stimuli evoke both ATP-mediated excitatory junction potentials (EJPs) and a slow noradrenaline (NA)-mediated depolarization (NAD). Here we have investigated the contribution of α 1- and α 2-adrenoceptors to the NAD. The α 1-adrenoceptor antagonist, prazosin (0.1 μM), and the α 2-antagonist, rauwolscine (1 μM), reduced the amplitude of the NAD and in combination these agents virtually abolished the NAD. The K ATP channel blocker, glibenclamide (10 μM) abolished the α 2-adrenoceptor-mediated component of the NAD, indicating that activation of these receptors produces closure of K ATP channels. The α 1-adrenoceptor-mediated component of the NAD was increased in amplitude by glibenclamide. Changes in membrane conductance were monitored by measuring the time constant of decay of EJPs (τEJP). The τEJP was increased during α 1-adrenoceptor-mediated depolarization, indicating a decrease in membrane conductance; i.e. closure of K + channels. Broad-spectrum K + channel blockers (tetraethylammonium, 4-aminopyridine, Ba 2+) and the TASK-1 K + channel blocker, anandamide (10 μM), did not reduce the α 1-adrenoceptor-mediated NAD. The α 1-adrenoceptor-mediated NAD was unaffected by the Cl − channel blockers, 9-anthracene carboxylic acid (100 μM) and niflumic acid (10 μM) or by the non-selective cation channel blocker, SKF 96365 (10 μM). These findings indicate that the NAD is produced by activation of both α 1-and α 2-adrenoceptors. The α 2-adrenoceptor-mediated component is produced by closure of K ATP channels whereas the α 1-adrenoceptor-mediated component is most likely mediated by closure of another type of K + channel.

Contamination of Membrane-Electrode Assemblies by Ammonia in Polymer Electrolyte Fuel Cells

Experimental and computational investigation of contamination of polymer electrolyte fuel cell (PEFC) membranes and catalyst layers with ammonia (NH3) is reported. Cyclic voltammetry (CV) scans and electrochemical impedance spectroscopy (EIS) analyses show that trace amounts of ammonia can significantly contaminate both the polymer electrolyte membrane (PEM) and the catalyst layers. The results show that the catalyst layer contamination can be reversed under certain conditions, while the membrane recovery tends to be much slower, and permanent effects of ammonia contamination is observed. A model is also developed to predict the degradation of the cell performance due to the decrease in membrane conductivity. The model incorporates Nernst-Planck equations to represent the multi-component cationic transport across the membrane, which helps differentiate between the degradation due to internal resistance and contamination of the catalyst layers. Mechanisms of contamination of the polymer electrolyte and catalyst layers, and performance degradation of the PEFC are also postulated.

Transient removal of alkaline zones after excitation of Chara cells is associated with inactivation of high conductance in the plasmalemma

The action potential (AP) of excitable plant cells is a multifunctional physiological signal. Its generation in characean algae suppresses the pH banding for 15–30 min and enhances the heterogeneity of spatial distribution of photosynthetic activity. This suppression is largely due to the cessation of H+ influx (OH– efflux) in the alkaline cell regions. Measurements of local pH and membrane conductance in individual space-clamped alkaline zones (small cell areas bathed in an isolated pool of external medium) showed that the AP generation is followed by the transient disappearance of alkaline zone in parallel with a large decrease in membrane conductance. These changes, specific to alkaline zones, were only observed under continuous illumination following a relaxation period of at least 15 min after previous excitation. The excitation of dark-adapted cells produced no conductance changes in the post-excitation period. The results indicate that the origin of alkaline zones in characean cells is not due to operation of electroneutral H+/HCO3– symport or OH–/HCO3– antiport. It is concluded that the membrane excitation is associated with inactivation of plasmalemma high conductance in the alkaline cell regions.

Acid-sensitive TASK-like K+ conductances contribute to resting membrane potential and to orexin-induced membrane depolarization in rat thalamic paraventricular nucleus neurons

Orexin (hypocretin) peptides are known to depolarize rat thalamic paraventricular nucleus (PVT) neurons by suppression of one or more undefined potassium conductances. Here, we investigated a contribution of TWIK-related acid-sensitive K+ (TASK) channels to the resting membrane potential and orexin-induced depolarization of PVT neurons, using patch clamp recording techniques in brain slice preparations. Upon exposure to an acidic (pH 6.3) extracellular solution, PVT neurons displayed membrane depolarization. Under voltage-clamp and in the presence of tetrodotoxin (TTX, 0.5 μM), low pH solutions induced an inward shift in baseline membrane current, accompanied by a net decrease in membrane conductance, reversing close to the potassium equilibrium potential. By contrast, exposure to alkaline (pH 8.3) solutions resulted in membrane hyperpolarization, induced an outward shift in baseline membrane current and an increase in net conductance that reversed close to the potassium equilibrium potential. A local anesthetic bupivacaine (20–40 μM) and the endocannabinoid anandamide (5–10 μM) mimicked the effects of the acidic solution. Exposure to the volatile anesthetic isoflurane (0.2–0.5 mM) induced changes in resting membrane potential, baseline current and membrane conductance similar to those caused by the alkaline solution. Although responsiveness to orexins was preserved under each of the above conditions, the amplitude of the orexin B (0.5 μM)-induced inward current was depressed in the acidic solution and in the presence of anandamide, remained largely unchanged in the alkaline solution, and was enhanced by isoflurane when compared with that in normal artificial cerebrospinal solution. We conclude that pH-sensitive potassium channels, TASK-1 and TASK-3 channels, contribute substantially to the resting membrane conductance(s) and excitability in PVT neurons. The observations that orexin-induced currents were affected by putative TASK-specific drugs in a manner predictable from their effects on TASK channels also suggest that the orexin-induced excitation in PVT neurons is mediated by closure of TASK channels.

Orexin peptides enhance median preoptic nucleus neuronal excitability via postsynaptic membrane depolarization and enhancement of glutamatergic afferents

Subpopulations of neurons in the median preoptic nucleus (MnPO) located within the lamina terminalis contribute to thermoregulatory, cardiovascular and hydromineral homeostasis, and sleep-promotion. MnPO is innervated by lateral hypothalamic neurons that synthesize and secrete the arousal-promoting and excitatory orexin (hypocretin) neuropeptides. To evaluate the hypothesis that orexins modulate the excitability of MnPO neurons, we used patch-clamp recording techniques applied in rat brain slice preparations to assess the effects of exogenously applied orexin A and orexin B peptides on their intrinsic and synaptic properties. Whole cell recordings under current-clamp mode revealed that 11/15 tested MnPO neurons responded similarly to either orexin A or B (500–1000 nM) with a slowly rising, prolonged (10–15 min) and reversible membrane depolarization. Under voltage-clamp mode, orexin applications induced a tetrodotoxin-resistant inward current of −7.2±1.6 pA, indicating a direct (postsynaptic) activation, with a time course similar to the observed membrane depolarization. The orexin-induced responses in 4/7 neurons were associated with a significant decrease in membrane conductance and the net orexin-induced current that reversed at −99±5 mV, suggesting closure of potassium channels. Orexins did not attenuate the properties of excitatory ( n=4) or inhibitory ( n=7) postsynaptic currents evoked by subfornical organ stimulation. By contrast, orexins applications induce a significant increase in both frequency and amplitude of spontaneous glutamatergic postsynaptic currents (5/7 cells) but had no influence on spontaneous GABAergic currents (6/6 cells). Thus, in addition to a direct postsynaptic receptor-mediated excitation, orexins can also increase the excitability of MnPO neurons via increasing their excitatory inputs, presumably through an orexin receptor–mediated excitation of local glutamatergic neurons whose axons project to MnPO neurons.

Estradiol modulation of phenylephrine-induced excitatory responses in ventromedial hypothalamic neurons of female rats

Estrogens act within the ventromedial nucleus of the hypothalamus (VMN) to facilitate lordosis behavior. Estradiol treatment in vivo induces alpha(1b)-adrenoreceptor mRNA and increases the density of alpha(1B)-adrenoreceptor binding in the hypothalamus. Activation of hypothalamic alpha(1)-adrenoceptors also facilitates estrogen-dependent lordosis. To investigate the cellular mechanisms of adrenergic effects on VMN neurons, whole-cell patch-clamp recordings were carried out on hypothalamic slices from control and estradiol-treated female rats. In control slices, bath application of the alpha(1)-agonist phenylephrine (PHE; 10 microM) depolarized 10 of 25 neurons (40%), hyperpolarized three neurons (12%), and had no effect on 12 neurons (48%). The depolarization was associated with decreased membrane conductance, and this current had a reversal potential close to the K(+) equilibrium potential. The alpha(1b)-receptor antagonist chloroethylclonidine (10 microM) blocked the depolarization produced by PHE in all cells. From estradiol-treated rats, significantly more neurons in slices depolarized (71%) and fewer neurons showed no response (17%) to PHE. PHE-induced depolarizations were significantly attenuated with 4-aminopyridine (5 mM) but unaffected by tetraethylammonium chloride (20 mM) or blockers of Na(+) and Ca(2+) channels. These data indicate that alpha(1)-adrenoceptors depolarize VMN neurons by reducing membrane conductance for K(+). Estradiol amplifies alpha(1b)-adrenergic signaling by increasing the proportion of VMN neurons that respond to stimulation of alpha(1b)-adrenergic receptors, which is expected in turn to promote lordosis.

Cholecystokinin B-Type Receptors Mediate a G-Protein-Dependent Depolarizing Action of Sulphated Cholecystokinin Ocatapeptide (CCK-8s) on Rodent Neonatal Spinal Ventral Horn Neurons

Reports of cholecystokinin (CCK) binding and expression of CCK receptors in neonatal rodent spinal cord suggest that CCK may influence neuronal excitability. In patch-clamp recordings from 19/21 ventral horn motoneurons in neonatal (PN 5-12 days) rat spinal cord slices, we noted a slowly rising and prolonged membrane depolarization induced by bath-applied sulfated CCK octapeptide (CCK-8s; 1 microM), blockable by the CCK B receptor antagonist L-365,260 (1 microM). Responses to nonsulfated CCK-8 or CCK-4 were significantly weaker. Under voltage clamp (V H -65 mV), 22/24 motoneurons displayed a CCK-8s-induced tetrodotoxin-resistant inward current [peak: -136 +/- 28 pA] with a similar time course, mediated via reduction in a potassium conductance. In 29/31 unidentified neurons, CCK-8s induced a significantly smaller inward current (peak: -42.8 +/- 5.6 pA), and I-V plots revealed either membrane conductance decrease with net inward current reversal at 101.3 +/- 4.4 mV (n = 16), membrane conductance increase with net current reversing at 36.1 +/- 3.8 mV (n = 4), or parallel shift (n = 9). Intracellular GTP-gamma-S significantly prolonged the effect of CCK-8s (n = 6), whereas GDP-beta-S significantly reduced the CCK-8s response (n = 6). Peak inward currents were significantly reduced after 5-min perfusion with N-ethylmaleimide. In isolated neonatal mouse spinal cord preparations, CCK-8s (30-300 nM) increased the amplitude and discharge of spontaneous depolarizations recorded from lumbosacral ventral roots. These observations imply functional postsynaptic G-protein-coupled CCK B receptors are prevalent in neonatal rodent spinal cord.