bluuebunny/crossref_metadata_2025 huggingface dataset:
Spontaneous motoneuronal activity mediated by glycine and GABA in the spinal cord of rat fetuses in vitro.
Authors: Nishimaru H , Iizuka M , Ozaki S , Kudo N
Published: December 2014

1. Spontaneous motoneuronal activity was monitored from the lumbar ventral roots in an isolated spinal cord preparation from rat fetuses at embryonic days (E) 13.5‐18.5. 2. Spontaneous bursts that were synchronized in both left and right ventral roots were observed periodically (mean interval, 1.5‐2.6 min) from E14.5 to 17.5. This activity was abolished in Ca(2+)‐free saline or by application of tetrodotoxin (1 microM), indicating that it was synaptically mediated. 3. The glutamate receptor blocker kynurenate (4 mM) failed to block spontaneous bursts at E14.5‐15.5, though it completely abolished them at E17.5. The glycine receptor antagonist strychnine (10 microM) completely blocked spontaneous bursts at E14.5‐15.5. Bicuculline, a GABAA receptor antagonist, reduced the amplitude of the spontaneous bursts. 4. At E15.5, a brief application of glycine (250 microM to 2 mM) evoked excitatory responses resembling the spontaneous bursts in both time course and amplitude. Such glycine‐induced responses were not observed under Ca(2+)‐free conditions, suggesting that they were synaptically evoked. These synaptic responses were not blocked by kynurenate (4 mM), but they were abolished by strychnine (10 microM). 5. It is concluded that glycine and GABA generate the earliest spontaneous motor activity of the fetus and function transiently as excitatory transmitters in the embryonic spinal cord.
Localizing quantal currents along frog neuromuscular junctions.
Authors: Van der Kloot W , Naves L A
Published: December 2014

1. We spatially localized the origins of quantal currents by recording simultaneously with two intracellular electrodes and employing the prediction of the one‐dimensional cable equations that the time integrals of the resulting voltage changes fall off exponentially with distance. 2. Miniature endplate potentials (MEPPs) were more frequent near the centre of the endplate. In contrast to some work using other methods, we did not find MEPPs originating at the margins of the endplate to be strikingly smaller. 3. Spontaneous MEPPs and uniquantal endplate potentials (EPPs) were released over the same length of endplate and with the same relative probabilities at different regions. 4. Nicotinic agonists decreased evoked quantal output, but did not change the length over which uniquantal EPPs were generated. We conclude they do not block nerve conduction in the terminals. 5. Data sets were obtained with an extracellular electrode and two intracellular electrodes. The extracellular electrode was invariably near the centre of the region in which congruous MEPPs appeared to be generated. However, the range in the calculated positions of the synchronous MEPPs was as long as 0.8 mm. Therefore, it may be possible that extracellular electrodes have a longer recording range than commonly assumed.
Potassium channel dysfunction in hypothalamic glucose‐receptive neurones of obese Zucker rats.
Authors: Rowe I C , Boden P R , Ashford M L
Published: December 2014

1. We have shown, using intracellular and cell‐attached recordings, that glucose‐receptive (GR) neurones of obese Zucker rats exhibit abnormal electrophysiological responses to changes in extracellular glucose concentration, whereas GR neurones of lean Zucker and control rats respond normally. 2. In inside‐out recordings from obese rat GR neurones it was shown that the 150 pS ATP‐sensitive K+ (KATP) and the 160 pS calcium‐activated K+ (KCa) channels were absent, whereas both were present in GR neurones of lean Zucker and control rats. 3. The potassium channel most frequently observed in inside‐out patches from obese GR neurones was characterized by a conductance of 213 pS, was activated by raising internal calcium and inhibited by application of internal ATP. This channel (which we have termed Kfa) was not observed in lean or control rat GR neurones. 4. Tolbutamide (100 microM) was found to induce no effect or to elicit a small depolarization of obese rat GR neurones in the absence of glucose, in contrast to its clear excitatory actions on control or lean Zucker GR neurones. 5. Intracellular, cell‐attached and inside‐out recordings from obese rat non‐GR neurones showed that there was no alteration in their membrane properties or firing characteristics or in the characteristics of the large‐conductance calcium‐activated K+ channel (KCa) present in these neurones as compared with lean and control rats. 6. It is concluded that the Kfa channel is specific to GR neurones of obese Zucker rats and that the presence of this channel coupled with the absence of KATP and KCa channels results in the abnormal glucose‐sensing response of these neurones.
Modulation of the muscarinic K+ channel by P2‐purinoceptors in guinea‐pig atrial myocytes.
Authors: Matsuura H , Ehara T
Published: December 2014

1. Activation of muscarinic K+ (KACh) channels by P2‐purinergic agonists, such as ATP, decreases monotonically in the continued presence of agonist. We investigated the mechanisms underlying this process of decline in guinea‐pig atrial myocytes using the patch‐clamp technique. 2. External ATP reversibly depressed the acetylcholine (ACh, 5.5‐11 microM)‐induced KACh current in a concentration‐dependent manner with a half‐maximal inhibitory concentration (IC50) of 5.4 microM. 3. External ATP irreversibly reduced guanosine‐5'‐O‐(3‐thiotriphosphate) (GTP gamma S)‐induced KACh current both in control and pertussis toxin (PTX)‐pretreated cells, suggesting (i) that the ATP‐induced inhibition of KACh current occurred at some step(s) downstream from the activation of the PTX‐sensitive G protein, GK, and (ii) that a PTX‐insensitive G protein was involved in the signal transduction pathway. 4. The potency order of ATP analogues in reducing KACh current was ATP > or = 2‐methylthio‐ATP > or = alpha, beta‐methylene‐ATP, indicating involvement of a P2Y‐type purinoceptor. 5. In the cell‐attached patch recording, ATP (100 microM) applied to the bath solution reduced the activity of the KACh channels activated by ACh in the pipette, in two out of eight experiments, suggesting the possible involvement of cytosolic second messengers in the inhibition of KACh channels. 6. The ATP‐induced reduction of KACh current was not affected by a protein kinase C inhibitor, 1‐(5‐isoquinolinesulphonyl)‐2‐methylpiperazine dihydrochloride (H‐7), suggesting that this response was not mediated by the activation of protein kinase C. 7. These results demonstrate that, in addition to the membrane‐delimited activation through GK, external ATP causes an inhibition of the KACh channel probably by activating a PTX‐insensitive G protein and cytosolic second messenger(s), which may underlie the monotonic decrease of the ATP‐activated KACh current.
Coherent inspiratory oscillation of cranial nerve discharges in perfused neonatal cat brainstem in vitro.
Authors: Kato F , Morin-Surun M P , Denavit-Saubié M
Published: December 2014

1. To understand the neural organization of respiratory movement control and its developmental transformation, we studied the temporal characteristics of inspiratory activities, especially nerve‐to‐nerve short‐term synchronization, in an in vitro preparation of the isolated, perfused brainstem of kittens aged 0‐14 days (postnatal day (P) 0‐14). 2. In the inspiratory discharges of facial, vagus, glossopharyngeal and hypoglossal nerves, a stable oscillation with a period of 30‐40 ms (i.e. approximately 30 Hz) was observed in all preparations examined. In addition, we demonstrated that this oscillation presents a strong short‐term synchrony between distinct inspiratory nerves. This nerve‐to‐nerve synchronization was already apparent at approximately 12 h after birth. The degree of synchronization as evaluated by coherence spectral analysis was larger than 0.85 in all cases at any age examined. 3. This nerve‐to‐nerve coherence was not affected by changes in temperature (28‐36 degrees C), whereas respiratory rate, oscillation frequency and oscillation amplitude as estimated by power spectral analysis were highly temperature sensitive. 4. The nerve‐to‐nerve synchronization, as well as the approximately 30 Hz oscillation, remained unchanged after a pontomedullary transection, indicating that the medullary network, completely isolated from other structures and afferents, is sufficient to produce both fast oscillation and nerve‐to‐nerve synchronization. 5. Based on these observations in vitro, we conclude that nerve‐to‐nerve coherent inspiratory oscillation generated in the brainstem is already functional early in life.
Release of Ca2+ is the crucial step for the potentiation of IPSCs in the cultured cerebellar Purkinje cells of the rat.
Authors: Hashimoto T , Ishii T , Ohmori H
Published: December 2014

1. Spontaneous inhibitory postsynaptic currents (IPSCs) and evoked IPSCs were recorded by a whole‐cell patch‐recording technique from cultured Purkinje cells of the rat. The size of spontaneous IPSCs, after a train of depolarizing pulses was applied to the Purkinje cells, increased to 163 +/‐ 6% (mean +/‐ S.E.M., n = 7 cells) of the control levels measured before the stimulus train. 2. The GABAergic postsynaptic currents were recorded under voltage clamp from the synapse formed between two Purkinje cells. These IPSCs increased to 218 +/‐ 31% (n = 4) of control levels after depolarizing stimulation was applied to the postsynaptic Purkinje cells. Size‐increased IPSCs were observed as long as recording continued and the phenomena will be called potentiation in this paper. 3. Intracellular application of Ruthenium Red (20 microM) did not block the potentiation of spontaneous IPSCs induced by the depolarizing stimulus (165 +/‐ 9%, n =6), but heparin (2 mg ml‐1) partially blocked the potentiation (123 +/‐ 10%, n = 6). Heparin applied together with Ruthenium Red (20 microM) blocked potentiation completely (96 +/‐ 5%, n = 8) at concentrations higher than 1 mg ml‐1. 4. Intracellular free calcium concentrations ([Ca2+]i) was monitored as the ratio of fura‐2 fluorescences excited at 340 and 380 nm. In control cells, [Ca2+]i was increased by each depolarizing pulse. When Purkinje cells were dialysed with heparin or heparin with Ruthenium Red, the rise in [Ca2+]i was suppressed. 5. Bath application of thapsigargin (1 microM) blocked the potentiation (99 +/‐ 2%, n = 4) and suppressed the rise in [Ca2+]i. 6. When 30 mM BAPTA was applied intracellularly, a train of depolarizing pulses failed to induce potentiation of IPSCs and failed to raise [Ca2+]i. The results from points 3‐6 suggest that the increase in [Ca2+]i, most probably coupled with the release from intracellular stores especially from the inositol trisphosphate (IP3)‐sensitive stores, is crucial for the potentiation of IPSCs. 7. Bath application of a metabotropic glutamate receptor activator (t‐ACPD, 200 microM) increased both the amplitude and frequency of spontaneous IPSCs and increased the [Ca2+]i slightly in dendrites. The inward current induced by the puff‐applied GABA (2 microM) was increased, after t‐ACPD application, to 186 +/‐ 36% of the control level (n = 3). Bath application of quisqualate (2 microM) caused a rapid increase in [Ca2+]i in dendrites and in the cell body and increased both the amplitude and frequency of spontaneous IPSCs. 8. The bath application of an inhibitor of phospholipase C (PLC), U73122 (1 microM), suppressed a rise in [Ca2+]i and blocked the potentiation (106 +/‐ 3%, n = 5). The inactive form, U73343 (1 microM), did not affect the potentiation (151 +/‐ 11%, n = 7) or the rise in [Ca2+]i. These observations suggest a possible involvement of the mechanism of Ca2+ activation of PLC and the IP3‐induced Ca2+ release in the induction of IPSC potentiation in Purkinje cells.
A slowly activating voltage‐dependent K+ current in rat pituitary nerve terminals.
Authors: Kilic G , Stolpe A , Lindau M
Published: December 2014

1. A novel slowly activating voltage‐dependent K+ current was observed in isolated nerve terminals from rat neurohypophysis using the whole‐cell configuration of the patch‐clamp technique. 2. The activation kinetics of the slow current could be fitted assuming Hodgkin‐‐Huxley‐type kinetics, an exponential, n, of 1.3 and activation time constants decreasing from 4 s at ‐50 mV to 0.7s at +40 mV. 3. A positive shift of reversal potential was observed when [K+] was increased in the bath solution. The current is carried mainly but not exclusively by K+ ions. 4. When intracellular free [Mg2+] was low (approximately 60 microM), average current density was 74 pA pF‐1 at membrane potentials around 0 mV. In 83% of nerve terminals current amplitude was > 10 pA pF‐1. 5. The slow current was never observed when the pipette contained 4.6 mM free Mg2+. At a physiological level of free Mg2+ (0.5 mM) the average current density was 16 pA pF‐1. 6. When nerve terminals were analysed after patch‐clamp experiments for vasopressin content by immunodetection, no difference in current amplitude was found between the terminals containing vasopressin and all analysed terminals. 7. The voltage dependence of activation was fitted by a Boltzmann equation giving a half‐activation potential of ‐37 mV and a slope factor of about 9 mV. 8. Tail current deactivation kinetics was biexponential with time constants of 0.12 and 1.5s. Kinetics was dependent on the duration of the activating pulse. 9. Noise analysis of the slow current indicated a single‐channel current of 0.33 pA at +6 mV, corresponding to a single‐channel conductance of 4.3 pS. 10. This is the first demonstration of a current similar to the slow K+ current, IKs, in a neurone, suggesting that a protein similar to the IKs‐inducing channel protein IsK (minK) may be present in peptidergic nerve terminals. 11. The activation properties are consistent with a role of the slow current in inhibition of excitability, at least at the level of the nerve terminal.
The source of physiologically stimulated glutamate efflux from the striatum of conscious rats.
Authors: Miele M , Boutelle M G , Fillenz M
Published: December 2014

1. Glutamate in the extracellular compartment of the striatum of freely moving rats was monitored at 5 min intervals using microdialysis and an enzyme‐based assay. 2. Basal levels of dialysate glutamate were 3.6 +/‐ 0.5 microM. Local infusion through the dialysis probe of tetrodotoxin (TTX), cadmium chloride or magnesium chloride produced no reduction in basal levels of glutamate; with the latter two there was, instead, an increase. 3. Neuronal activation stimulated by induced grooming was accompanied by an increase in total glutamate efflux of 47.5 +/‐ 25.0% above basal level; this increase was not reduced by local infusion of TTX. 4. We propose that the TTX‐insensitive release of glutamate in response to physiological stimulation is derived from glial cells and is a Ca(2+)‐dependent mechanism triggered by a receptor‐mediated release of Ca2+ from internal stores that spreads through the network of astrocytes.
Effects of stimulation of phrenic afferents on cervical respiratory interneurones and phrenic motoneurones in cats.
Authors: Iscoe S , Duffin J
Published: December 2014

1. In ten decerebrate, paralysed and ventilated cats, we tested the hypothesis that cervical (C5) respiratory interneurones mediate inhibition of phrenic motoneurone activity resulting from single shocks to the phrenic nerve. 2. Stimulus intensities sufficient to activate all afferents elicited (latency, 4.0 +/‐ 0.9 ms, mean +/‐ S.D.) a graded suppression of ipsilateral, but not contralateral (five of seven cats) phrenic nerve activity lasting, in six of seven cats, more than 70 ms and interrupted by a brief (approximately 6‐18 ms duration) excitation at latencies between 7 and 30 ms. 3. In twenty‐five ipsilateral motoneurones, peristimulus time average of the membrane potentials (‐61 +/‐ 10 mV) showed no effect in eleven; of the fourteen that responded, ten had initial EPSPs (latency, 17.6 +/‐ 3.0 ms) and four initial IPSPs (latencies, 2.25‐4.3 ms). Only one motoneurone had both. No responses with latencies > 60 ms were observed. 4. Peristimulus time averages of extracellular activity of thirty ipsilateral interneurones, twenty‐five firing in inspiration (I) and five in expiration (E), showed diverse responses. The initial response of I interneurones was an excitation in eleven, a suppression of activity in nine, and no response in five. Latencies of excitations ranged from 2 to 36.5 ms (median, 14 ms) with durations ranging from 2 to 7 ms (mean, 4.4 +/‐ 1.6 ms). Latencies of suppression of activity ranged from 2 to 29 ms (median, 10 ms). Two E interneurones were excited (latencies, 11 and 15 ms; durations, 3.5 and 2 ms), two inhibited (latencies, 2 and 12 ms; durations, > 40 and 17 ms, respectively), and one did not respond. 5. In nine interneurones (seven I, two E), peristimulus time averages of the membrane potentials (mean, ‐62 +/‐ 14 mV) revealed no effect on three (all I). Of the six that responded, four (three I) had initial IPSPs, two (one I, one E) initial EPSPs. EPSPs had latencies of 11.5 (I interneurone) and 22 ms (E interneurone); the latencies of the IPSPs were 2.75, 3.20, and 2.3 ms for the I interneurones and 15.9 ms for the E interneurone). No responses with latencies > 30 ms were observed. 6. The diverse responses of cervical respiratory interneurones indicates that they do not mediate the prolonged suppression of ipsilateral phrenic activity elicited by stimulation of phrenic afferents. The suppression may result from activation of normally quiescent inhibitory interneurones or from presynaptic inhibition.
Role of ganglionic cotransmission in sympathetic control of the isolated bullfrog aorta.
Authors: Thorne R , Horn J P
Published: December 2014

1. The relation between preganglionic activity and arterial tone was studied in preparations of bullfrog lumbar sympathetic ganglia 7‐10 and the dorsal aorta. 2. Two or more stimuli evoked contractions when applied to the preganglionic C, but not the B pathway. Contractions were blocked when transmission in ganglia 9 and 10 was disrupted by cutting the sympathetic chain or adding (+)‐tubocurarine. Contractions were antagonized by postganglionic action of guanethidine, but not by phentolamine or suramin. 3. Aortic responses to short trains (10‐100 stimuli) were half‐maximal at 0.3‐0.5 Hz, saturated near 1 Hz and had a minimum latency of 8.9 s. By contrast, responses to 300 stimuli were half‐maximal at 1 Hz and became 2.5‐fold larger at 10 Hz. 4. Exogenous luteinizing hormone releasing hormone (LHRH) potentiated preganglionically evoked contractions. Endogenous LHRH mediated contractions evoked by 10 Hz stimulation in (+)‐tubocurarine. These responses had a longer latency than in normal Ringer solution and were blocked by [D‐pGlu1, D‐Phe2, D‐Trp3.6]‐LHRH. The LHRH antagonist did not alter contractions evoked by continuous stimulation in normal Ringer solution or by bursts of stimuli in hexamethonium. 5. Exogenous neuropeptide Y (NPY) potentiated neurogenic contractions and responses to adrenaline. Benextramine blocked contractions produced by nerve stimulation, adrenaline and NPY, but not ATP. 6. The results show that contractions of the isolated aorta are tuned to physiological frequencies of activity in sympathetic C neurones. Peptidergic cotransmission in the ganglia can increase arterial tension, but not during synchronous activation of primary nicotinic synapses. It is suggested that the physiological role of LHRH arises from interactions with subthreshold nicotinic EPSPs and that postganglionic release of NPY shifts frequency tuning of the circuit during prolonged activity.
Similarity of ATP‐dependent K+ channels in skeletal muscle fibres from normal and mutant mdx mice.
Authors: Allard B , Rougier O
Published: December 2014

1. ATP‐dependent K+ (KATP) channels were studied in fibres isolated from flexor digitorum brevis and interosseal skeletal muscles of normal and mutant mdx mice using the patch clamp technique in the presence of asymmetrical K+ concentrations (5 mM K+ in the pipette and in vivo intracellular [K+] or 145 mM K+ at the cytoplasmic face). 2. In cell‐attached patches from mdx muscle fibres bathed in K(+)‐rich solution, cell poisoning with fluorodinitrobenzene induced partially reversible opening of channels carrying an outward current of an amplitude of 1.2 pA at 0 mV. Exposure of fibres to the K+ channel opener cromakalim led to opening of the same type of channel. These channels were assumed to be KATP channels. 3. On excision of inside‐out patches from mdx muscle fibres, in the absence of intracellular ATP, KATP channels were active: they carried a unitary outward current of 1.6 pA at 0 mV and were inhibited by intracellular ATP and glibenclamide. The number of KATP channels per patch was not significantly different in muscles from normal and mdx mice. 4. In inside‐out patches, in the presence of 1 mM intracellular Mg2+, slope conductances of 21 and 20.3 pS were found for KATP channels in normal and mdx muscle, respectively. In the absence of Mg2+, slope conductances of KATP channels were 31.3 and 32 pS in normal and mdx muscle, respectively and KATP channel activity was augmented in mdx muscle in the same way as in normal muscle. Activity of the same KATP channel was observed in extensor digitorum longus muscle from normal and mdx mice. 5. In inside‐out patches held at 0 mV, the relationship between KATP channel activity and intracellular ATP was described by a Hill equation: Ki values were 23 and 21 microM and Hill coefficients were 1.8 and 1.9 in normal and mdx muscle, respectively. 6. These results indicate that the distribution, the conductance properties and ATP sensitivity of KATP channels do not differ in normal and in mdx mouse skeletal muscle.
Calcium‐activated chloride channels in bovine pulmonary artery endothelial cells.
Authors: Nilius B , Prenen J , Szücs G , Wei L , Tanzi F , Voets T , Droogmans G
Published: December 2014

1. We characterized Ca(2+)‐activated Cl‐ currents in calf pulmonary artery endothelial (CPAE) cells by using a combined patch clamp and fura‐2 microfluorescence technique to simultaneously measure ionic currents and the intracellular Ca2+ concentration, [Ca2+]i. 2. Various procedures that increased [Ca2+]i, such as stimulation with ATP or ionomycin, or loading the cells with Ca2+ via the patch pipette, activated a strongly outwardly rectifying current with a reversal potential close to the Cl‐ equilibrium potential. Changing the extracellular Cl‐ concentration shifted this reversal potential as predicted for a Cl‐ current. Buffering Ca2+ rises with BAPTA prevented ATP from activating the current. 3. Ca(2+)‐activated Cl‐ currents could be distinguished from volume‐activated Cl‐ currents, which were sometimes coactivated in the same cell. The latter showed much less outward rectification, their activation was voltage independent, and they could be inhibited by exposing the cells to hypertonic solutions. 4. The permeability ratio for the Ca(2+)‐activated conductance of the anions iodide:chloride: gluconate was 1.71 +/‐ 0.06:1:0.39 +/‐ 0.03 (n = 12). 5. This Ca(2+)‐activated Cl‐ current, ICl, Ca, inactivated rapidly at negative potentials and activated slowly at positive potentials. Outward tail currents were slowly decaying, while inward tail currents decayed much faster. 6. 4,4'‐Diisothiocyanatostilbene‐2,2'‐disulphonic‐acid (DIDS) and niflumic acid inhibited Icl,Ca in a voltage‐dependent manner, i.e. they exerted a more potent block at positive potentials. The block by N‐phenylanthracilic acid (NPA), 5‐nitro‐2‐(3‐phenylpropylamino)‐benzoate (NPPB) and tamoxifen was voltage independent. Niflumic acid and tamoxifen were the most potent blockers. 7. The single‐channel conductance was 7.9 +/‐ 0.7 pS (n = 15) at 300 mM extracellular Cl‐. The channel open probability was high at positive potentials, but very small at negative potentials. 8. It is concluded that [Ca2+]i activates small‐conductance Cl‐ channels in endothelial cells, which coexist with the volume‐activated Cl‐ channels described previously.
Calcium‐activated chloride current in normal mouse sympathetic ganglion cells.
Authors: De Castro F , Geijo-Barrientos E , Gallego R
Published: December 2014

1. In rat sympathetic ganglion cells, axotomy induces the appearance of a depolarizing after‐potential (ADP) produced by a calcium‐activated chloride current. Here we report that this current is also present in normal sympathetic neurones from the mouse. 2. In an in vitro preparation of the superior cervical ganglion, an ADP was observed after spike firing in 50% of the cells studied with single‐electrode current‐ and voltage‐clamp techniques. 3. When the cells were voltage clamped at ‐50 mV in the presence of tetrodotoxin (TTX) and tetraethylammonium chloride (TEA), depolarizing jumps evoked inward calcium currents which were contaminated by outward chloride currents, followed by slowly decaying inward chloride tail currents. 4. The ADP and the inward tail currents disappeared when calcium was removed from the extracellular solution or when cadmium was added. 5. The reversal potential for the inward tail current was approximately ‐24 mV and was displaced in agreement with the Nernst equation for chloride when the extracellular NaCl was replaced by sucrose or sodium isethionate. The chloride channel blocker anthracene‐9‐carboxylic acid (9AC) inhibited both the ADP and the tail current. 6. Using intracellular injection of neurobiotin, we found that cells with shorter dendrites had larger ADPs. In axotomized ganglia practically all cells showed very pronounced ADPs. 7. We conclude that normal mouse sympathetic ganglion cells have a calcium‐activated chloride current that generates an ADP. The channels responsible for this current are probably located in the dendrites.
Synchronization of GABAergic interneuronal network in CA3 subfield of neonatal rat hippocampal slices.
Authors: Khazipov R , Leinekugel X , Khalilov I , Gaiarsa J L , Ben-Ari Y
Published: December 2014

1. Cell‐attached and whole‐cell recordings from interneurons localized in the stratum radiatum of the CA3 subfield (SR‐CA3) of neonatal (postnatal days 2‐5) rat hippocampal slices were performed to study their activity during the generation of GABAergic giant depolarizing potentials (GDPs) in CA3 pyramidal cells. 2. Dual recordings revealed that during the generation of GDPs in CA3 pyramidal cells, the interneurons fire bursts of spikes, on average 4.5 +/‐ 1.4 spikes per burst (cell‐attached mode). There bursts were induced by periodical large inward currents (interneuronal GDPs) recorded in whole‐cell mode. 3. Interneuronal GDPs revealed typical features of polysynaptic neuronal network‐driven events: they were blocked by TTX and by high divalent cation medium and they could be evoked in an all‐or‐none manner by electrical stimulation in different regions of the hippocampus. The network elements required for the generation of GDPs are present in local CA3 circuits since spontaneous GDPs were present in the isolated CA3 subfield of the hippocampal slice. 4. Interneuronal GDPs were mediated by GABAA and glutamate receptors, since: (i) their reversal potential strongly depended on [Cl‐]i; (ii) at the reversal potential of GABAA postsynaptic currents an inward component of GDPs was composed of events with the same kinetics as alpha‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (AMPA) receptor‐mediated EPSCs; and (iii) once GABAA receptors were blocked intracellularly by dialysis with F(‐)‐MgATP‐free solution, the remaining component of interneuronal GDPs reversed near 0 mV and rectified at membrane potentials more negative than ‐20 mV, suggesting an important contribution of NMDA receptors in addition to AMPA receptors. 5. In cell‐attached recordings from interneurons, electrical stimulation in the stratum radiatum evoked a burst of spikes that corresponded to evoked GDPs. Pharmacological study of this response revealed that excitation of SR‐CA3 interneurons during GDPs is determined by the co‐operative depolarizing actions mediated by GABAA and glutamate (AMPA and NMDA) receptors. Interestingly, after blockade of AMPA receptors, GABAA receptor‐mediated depolarization enabled the activation of NMDA receptors presumably via attenuation of their voltage‐dependent magnesium block. 6. It is concluded that synchronous activation of SR‐CA3 interneurons during generation of GDPs is mediated synaptically and is determined by the co‐operation of (i) excitatory GABAergic connections between interneurons and (ii) glutamatergic connections to interneurons originating presumably from the pyramidal cells.
Measurement of the distribution of anion exchange function in normal human red cells.
Authors: Raftos J E , Bookchin R M , Lew V L
Published: December 2014

1. The aim of the present work was to investigate cell‐to‐cell variation in anion exchange turnover in normal human red cells. Red cells permeabilized to protons and K+ dehydrate extremely rapidly by processes that are rate‐limited by the induced K+ permeability or by anion exchange turnover. Conditions were designed to render dehydration rate‐limited by anion exchange turnover. Cell‐to‐cell variation in anion exchange function could then be measured from the distribution of delay times required for dehydrating cells to attain resistance to haemolysis in a selected hypotonic medium. 2. Red cells were suspended at 10% haematocrit in a low‐K+ solution and, after a brief preincubation with 20 microM SITS at 4 degrees C, were warmed to 24 degrees C, and the protonophore CCCP was added (20 microM) followed 2 min later by valinomycin (60 microM). Delay times for cells to become resistant to lysis were measured from the instant of valinomycin addition by sampling suspension aliquots into thirty volumes of 35 mM NaCl. After centrifugation the per cent lysis was estimated by measuring the haemoglobin concentration in the supernatant. Typical median delay times with this standardized method were 4‐5 min. 3. The statistical parameters of the delay time distributions report the population spread in the transport function that was limiting to dehydration. In the absence of SITS and CCCP, dehydration was limited by the diffusional Cl‐ permeability (PCl). Delay time distributions for PCl‐ and anion exchange‐limited dehydration were measured in red cells from three normal donors. For both distributions, the coefficients of variation ranged between 13.0 and 15.2%, indicating a high degree of uniformity in PCl and anion exchange function among individual red cells.
Endothelin‐1 potently stimulates chloride secretion and inhibits Na(+)‐glucose absorption in human intestine in vitro.
Authors: Kuhn M , Fuchs M , Beck F X , Martin S , Jähne J , Klempnauer J , Kaever V , Rechkemmer G , Forssmann W G
Published: December 2014

1. Serosally added synthetic endothelin‐1 (ET‐1) increased short‐circuit current (Isc) across isolated muscle‐stripped human colonic mucosa in vitro. Bumetanide inhibited Isc responses, indicating that ET‐1 stimulates electrogenic Cl‐ secretion. 2. In isolated human jejunal mucosa, ET‐1 exhibited a concentration‐dependent dual action. At low concentrations it induced rapid increases in Isc and these were inhibited by bumetanide. At a higher concentration (0.1 microM), ET‐1 provoked a drastic and progressive decrease in Isc below the baseline value. 3. Pretreatment with phlorizin or omission of glucose from the Krebs‐Ringer solution at the apical (luminal) side of the jejunal mucosa prevented the decreases in Isc evoked by ET‐1, suggesting that the peptide inhibits the glucose‐coupled electrogenic Na+ absorption. Indeed, flux experiments with D‐[14C]glucose demonstrated that ET‐1 decreases jejunal glucose absorption by approximately 80% within 30 min. 4. Electron microprobe analyses of cryosections of human jejunum showed that ET‐1 (0.1 microM) evokes a significant decrease in intracellular Na+ concentrations of villus (not crypt) epithelial cells, suggesting that the peptide attenuates apical Na(+)‐glucose entry by reducing the activity of the Na(+)‐glucose cotransporter, SGLT1. 5. In the presence of tetrodotoxin (TTX), ET‐1‐induced Cl‐ secretion was significantly reduced, in both human jejunal and colonic mucosa. However, the inhibitory effect on jejunal Na(+)‐glucose absorption was not affected by TTX. 6. ET‐1 increases electrogenic Cl‐ secretion across human intestinal mucosa in vitro. This effect is mediated in part via the activation of enteric nerves. Responses of the human jejunal mucosa to high ET‐1 concentrations exhibit a second component, namely the rapid inhibition of electrogenic Na(+)‐glucose absorption, which might be mediated by an inhibition of the transport activity of SGLT1. This effect is independent from neuronal mediators. Our results suggest different cellular action sites for ET‐1 in human small and large intestine.
Possible role of atypical protein kinase C activated by arachidonic acid in Ca2+ sensitization of rabbit smooth muscle.
Authors: Gailly P , Gong M C , Somlyo A V , Somlyo A P
Published: December 2014

1. Diacylglycerol (DAG; 10 microM), an activator of conventional and novel protein kinases C (cPKCs and nPKCs), induced Ca2+ sensitization of force in isolated intact and alpha‐toxin‐permeabilized femoral artery (FA) and portal vein (PV), and increased the phosphorylation of myosin light chain (MLC20) at the same peptides phosphorylated by myosin light chain kinase. 2. Ca2+ sensitization by DAG was specifically inhibited by a pseudosubstrate peptide inhibitor of cPKCs (PKC alpha(22‐30) peptide; 50 microM). Similarly, GF 109203X (600 nM), an inhibitor of cPKCs and nPKCs, completely abolished Ca2+ sensitization by phorbol 12,13‐dibutyrate (PDBu; 1 microM). In contrast, Ca2+ sensitization induced by the alpha1‐adrenergic agonist phenylephrine (100 microM) was not inhibited by these inhibitors of cPKCs and nPKCs. 3. A pseudosubstrate peptide inhibitor of the atypical PKCs (aPKCs) PKC zeta(116‐124) (50 microM) significantly (about 50%) inhibited the Ca2+ sensitization of force and MLC20 phosphorylation induced by 100 microM phenylephrine and by 300 microM arachidonic acid, but not that by DAG (10 microM) or PDBu (1 microM). 4. A phospholipase A2 (PLA2) inhibitor, ONO‐RS‐082 (10 microM), abolished the release of arachidonic acid and partially (by 40%) inhibited the Ca2+ sensitization induced by phenylephrine in FA smooth muscle. This effect was not additive to the inhibition observed with the aPKC inhibitor peptide, suggesting that arachidonic acid and aPKCs exert their effects via the same pathway, probably through activation of aPKC(s) by arachidonic acid. 5. Western blot analysis with antibodies to aPKCs revealed aPKCs zeta, lambda (or iota) and an unidentified 64 kDa protein. The distribution (cytosolic and particulate) of these proteins was not affected by PDBu (1 microM). 6. Our results are consistent with a significant role for atypical (or related) PKCs through a PLA2‐arachidonic acid‐aPKC pathway in agonist‐induced Ca2+ sensitization, in parallel with a similar, but minor role of the DAG‐cPKC cascade. The inability of the combination of the two (aPKC and cPKC) inhibitors to completely eliminate Ca2+ sensitization also suggests the presence of a third, still unidentified, pathway of this mechanism.
Excitatory synaptic site heterogeneity during paired pulse plasticity in CA1 pyramidal cells in rat hippocampus in vitro.
Authors: Turner D A , Chen Y , Isaac J T , West M , Wheal H V
Published: December 2014

1. The properties of individual excitatory synaptic sites onto adult CA1 hippocampal neurons were investigated using paired pulse minimal stimulation and low noise whole‐cell recordings. Non‐NMDA receptor‐mediated synaptic responses were isolated using a pharmacological blockade of NMDA and GABAA receptors. Amongst the twenty‐five stationary ensembles there were twelve showing paired pulse potentiation, two showing paired pulse depression and eleven with no significant net change. The signal‐to‐noise ratio averaged 4.5:1. There was no correlation between the amplitude of the first and second responses after separation of failures: the percentage of failures averaged 33.6% for the conditioning pulse and 31.7% for the test pulse. 2. Site‐directed Bayesian statistical analysis was developed to predict the likely number of activated synapses, synaptic response amplitudes, probability of release and intrinsic variation at each individual synaptic site. Extensive simulations showed the usefulness of this model and defined appropriate parameters. These simulations demonstrated only small errors in estimating parameters of data sets with a small number of sites (< 10) and similar characteristics to the physiological data sets. 3. Physiological ensembles showed between one and three synaptic sites, which exhibited a wide range of values for release probability (0.03‐0.99), synaptic amplitudes (1.46‐16.8 pA; approximately 62% coefficient of variation between sites) and intrinsic variation over time (approximately 36%). Paired pulse plasticity occurred primarily from alterations in the release probabilities but a few ensembles also showed small changes in site amplitude. Initial release probability correlated negatively with the degree of paired pulse potentiation. Whilst it was possible to use simple assumptions regarding site homogeneity (such as required for a binomial process) for 48% (12 out of 25) of the data sets, the Bayesian analysis was necessary to reveal the complex changes and heterogeneity that occurred in the other 52% of the data sets. The Bayesian site analysis robustly indicated the presence of considerable site heterogeneity, significant intrinsic site variation over time and changes in parameters at individual synaptic sites with plasticity.
Functional improvement of damaged adult mouse muscle by implantation of primary myoblasts.
Authors: Irintchev A , Langer M , Zweyer M , Theisen R , Wernig A
Published: December 2014

1. Myoblasts from expanded primary cultures were implanted into cryodamaged soleus muscles of adult BALB/c mice. One to four months later isometric tension recordings were performed in vitro, and the male donor cells implanted into female hosts were traced on histological sections using a Y‐chromosome‐specific probe. The muscles were either mildly or severely cryodamaged, which led to reductions in tetanic muscle force to 33% (n = 9 muscles, 9 animals) and 70% (n = 11) of normal, respectively. Reduced forces resulted from deficits in regeneration of muscle tissue as judged from the reduced desmin‐positive cross‐sectional areas (34 and 66% of control, respectively). 2. Implantation of 10(6) myogenic cells into severely cryodamaged muscles more than doubled muscle tetanic force (to 70% of normal, n = 14), as well as specific force (to 66% of normal). Absolute and relative amount of desmin‐positive muscle cross‐sectional areas were significantly increased indicating improved microarchitecture and less fibrosis. Newly formed muscle tissue was fully innervated since the tetanic forces resulting from direct and indirect (nerve‐evoked) stimulation were equal. Endplates were found on numerous Y‐positive muscle fibres. 3. As judged from their position under basal laminae of muscle fibres and the expression of M‐cadherin, donor‐derived cells contributed to the pool of satellite cells on small‐ and large‐diameter muscle fibres. 4. Myoblast implantation after mild cryodamage and in undamaged muscles had little or no functional or structural effects; in both preparations only a few Y‐positive muscle nuclei were detected. It is concluded that myoblasts from expanded primary cultures‐unlike permanent cell lines‐significantly contribute to muscle regeneration only when previous muscle damage is extensive and loss of host satellite cells is severe.
A Physically Based, One-Dimensional Two-Fluid Model for Direct Contact Condensation of Steam Jets Submerged in Subcooled Water
Authors: Heinze David , Schulenberg Thomas , Behnke Lars
Published: December 2014

A simulation model for the direct contact condensation of steam in subcooled water is presented that allows determination of major parameters of the process, such as the jet penetration length. Entrainment of water by the steam jet is modeled based on the Kelvin–Helmholtz and Rayleigh–Taylor instability theories. Primary atomization due to acceleration of interfacial waves and secondary atomization due to aerodynamic forces account for the initial size of entrained droplets. The resulting steam-water two-phase flow is simulated based on a one-dimensional two-fluid model. An interfacial area transport equation is used to track changes of the interfacial area density due to droplet entrainment and steam condensation. Interfacial heat and mass transfer rates during condensation are calculated using the two-resistance model. The resulting two-phase flow equations constitute a system of ordinary differential equations, which is solved by means of the explicit Runge–Kutta–Fehlberg algorithm. The simulation results are in good qualitative agreement with published experimental data over a wide range of pool temperatures and mass flow rates.
Heat Sink With Stacked Multi-Layer Porous Media
Authors: Karunanithi Arun K. , Hassanipour Fatemeh
Published: December 2014

Previous studies have shown that stacked multi-layer mini-channels heat sinks with square or circular channels have advantages over traditional single layered channels in terms of both pressure drop and thermal resistance. In this work, porous media is used in the multi-layered stacked mini-channels instead of square or rectangular channels and the effect of the same on pressure drop and thermal performance is studied. Porosity scaling is done between the layers of porous media and is compared with unscaled stacked multilayer channel. Porosity scaling allows the porosity to vary from one layer to the next layer and could result in a lower pressure drop and better thermal performance.
Thermal Characterization of Multiple Micro-Jet Impingement Cooling Model
Authors: Husain Afzal , Kim Jun-Hee , Kim Kwang-Yong
Published: December 2014

The present study investigated thermal performance of silicon-based multiple micro-jet impingement cooling heat sink for thermal management of electronics. Three-dimensional numerical analysis was performed for steady incompressible laminar flow and conjugate heat transfer through a finite volume solver. A heat flux of 100 W/cm2 was applied at one side of the silicon substrate, while at the other side jet impingement system was designed. The jet plate was consisted of many jet holes whereas computational domain was simplified by utilizing symmetric boundary conditions along the flow as well as lateral directions. The effect of various design parameters, namely, jet diameter, jet pitch, standoff (distance from jet exit to impingement surface) etc., have been analyzed at jet Reynolds numbers 100, 200 and 300 under laminar flow conditions. In view of the low pumping powers available through micro-pumping systems, low flow rates were applied for the analysis. The cross-flow effects of the spent-flow were investigated for finding out optimum design parameters and flow conditions for the heat sink. The temperature distribution was discussed for various values of jet diameter, standoff and jet-to-jet spacing. While a moderate thermal resistance of the heat sink was obtained under laminar flow conditions, high performance can be achieved for higher flow-rate turbulent flow conditions at the expense of excessive pressure-drop which would be investigated in future studies.
Finite Element Volume Analysis of Propane Preheated Air Flame Passing Through a Minichannel
Authors: Darbandi Masoud , Ghafourizadeh Majid , Schneider Gerry E.
Published: December 2014

A hybrid finite-element-volume FEV method is extended to simulate turbulent non-premixed propane air preheated flame in a minichannel. We use a detailed kinetics scheme, i.e. GRI mechanism 3.0, and the flamelet model to perform the combustion modeling. The turbulence-chemistry interaction is taken into account in this flamelet modeling using presumed shape probability density functions PDFs. Considering an upwind-biased physics for the current reacting flow, we implement the physical influence upwinding scheme PIS to estimate the cell-face mixture fraction variance in this study. To close the turbulence closure, we employ the two-equation standard κ-ε turbulence model incorporated with suitable wall functions. Supposing an optically thin limit, it needs to take into account radiation effects of the most important radiating species in the current modeling. Despite facing with so many flame instabilities in such small size configuration, the current method performs suitably with proper convergence, and the encountered instabilities are damped out automatically. Comparing with the experimental measurements, the current extended method accurately predicts the flame structure in the minichannel configuration.
The spinless relativistic Yukawa problem
Authors: Lucha Wolfgang , Schöberl Franz F.
Published: December 2014

Noticing renewed or increasing interest in the possibility to describe semirelativistic bound states (of either spin-zero constituents or, upon confining oneself to spin-averaged features, constituents with nonzero spin) by means of the spinless Salpeter equation generalizing the Schrödinger equation toward incorporation of effects caused by relativistic kinematics, we revisit this problem for interactions between bound-state constituents of Yukawa shape, by recalling and applying several well-known tools enabling to constrain the resulting spectra.
RhoC Is an Unexpected Target of RhoGDI2 in Prevention of Lung Colonization of Bladder Cancer
Authors: Griner Erin M. , Dancik Garrett M. , Costello James C. , Owens Charles , Guin Sunny , Edwards Michael G. , Brautigan David L. , Theodorescu Dan
Published: December 2014

RhoGDI2 (ARHGDIB) suppresses metastasis in a variety of cancers but the mechanism is unclear, thus hampering development of human therapeutics. RhoGDI2 is a guanine nucleotide dissociation inhibitor (GDI) for the Rho family of GTPases thought to primarily bind to Rac1; however, Rac1 activation was not decreased by RhoGDI2 expression in bladder cancer cells. To better understand the GTPase-binding partners for RhoGDI2, a mass spectrometry–based proteomic approach was used in bladder cancer cells. As expected, endogenous RhoGDI2 coimmunoprecipitates with Rac1 and unexpectedly also with RhoC. Further analysis demonstrated that RhoGDI2 negatively regulates RhoC, as knockdown of RhoGDI2 increased RhoC activation in response to serum stimulation. Conversely, overexpression of RhoGDI2 decreased RhoC activation. RhoC promoted bladder cancer cell growth and invasion, as knockdown increased cell doubling time, decreased invasion through Matrigel, and decreased colony formation in soft agar. Importantly, RhoC knockdown reduced in vivo lung colonization by bladder cancer cells following tail vein injection in immunocompromised mice. Finally, unbiased transcriptome analysis revealed a set of genes regulated by RhoGDI2 overexpression and RhoC knockdown in bladder cancer cells.

Implications: RhoGDI2 suppresses bladder cancer metastatic colonization via negative regulation of RhoC activity, providing a rationale for the development of therapeutics that target RhoC signaling. Mol Cancer Res; 13(3); 483–92. ©2014 AACR.