bluuebunny/crossref_metadata_2025 huggingface dataset:
The actin filament disrupter cytochalasin D activates the recombinant cystic fibrosis transmembrane conductance regulator Cl‐ channel in mouse 3T3 fibroblasts.
Authors: Fischer H , Illek B , Machen T E
Published: December 2014

1. Cytochalasin D (CD; 5 microM) readily stimulated cystic fibrosis transmembrane conductance regulator (CFTR) Cl‐ channel activity in cell‐attached and whole‐cell patch recordings from 3T3 fibroblasts expressing recombinant CFTR but not in mock‐transfected cells. CD‐stimulated currents were indistinguishable from those evoked by forskolin stimulation. Kinetic analysis of CFTR gating showed identical channel behaviour independent of the agonist used. 2. To elucidate the mechanism of action of CD we tested its effects on cAMP, protein kinase A, and the CFTR itself during CD stimulation. In contrast to forskolin treatment, CD did not increase cellular cAMP content. 3. A direct interaction of CD with the CFTR was ruled out because CD showed no effect on CFTR in excised inside‐out patches. 4. CD effects were fully blocked when the cellular protein kinase A was inhibited by treatment of cells with RpcAMPS in cell‐attached patches or when protein kinase inhibitor peptide was dialysed into cells in whole‐cell experiments. 5. Addition of G‐actin to excised patches had no effects on CFTR. 6. We conclude that the stimulatory effect of CD is cAMP independent, but needs a functional protein kinase A in order to activate the CFTR. We propose that cytochalasin D activates CFTR by releasing a cellular inhibitor, e.g. a phosphatase, that is held in place by F‐actin.
Functional and non‐functional isoforms of the human muscle acetylcholine receptor.
Authors: Newland C F , Beeson D , Vincent A , Newsom‐Davis J
Published: December 2014

1. The properties of a recently identified isoform of the human muscle nicotinic acetylcholine receptor (AChR) alpha subunit (alpha +), which in muscle is expressed at similar levels to the alpha subunit, were investigated by both electrophysiological and biochemical approaches following expression in Xenopus laevis oocytes. The single‐channel properties of adult (alpha 2 beta delta epsilon) and fetal (alpha 2 beta delta gamma) forms of the human AChR were also investigated. 2. The mean burst duration of adult channels (4.1 +/‐ 0.3 ms, mean +/‐ S.E.M., n = 5) is half that of fetal channels (7.9 +/‐ 0.6 ms, n = 4), while the single‐channel conductance is larger (62.2 +/‐ 0.8 and 37.9 +/‐ 1.6 pS for adult and fetal channels, respectively), comparable to the developmental changes in single‐channel properties observed for other mammalian species. 3. In contrast to the alpha isoform, the alpha + subunit does not bind 125I‐labelled alpha‐bungarotoxin or monoclonal antibodies directed against the AChR ‘main immunogenic region’ (MIR), illustrating why the alpha + subunit was first detected through screening of cDNA libraries. 4. By using site‐directed mutagenesis to produce subunits that conferred different single‐channel conductances on the AChR, we demonstrate that the alpha + isoform is not integrated into functional AChRs. 5. The mutagenesis experiments also revealed that the two alpha subunits within an AChR pentamer are not equivalent within the pore lining region.
Synchronization between motor cortex and spinal motoneuronal pool during the performance of a maintained motor task in man.
Authors: Conway B A , Halliday D M , Farmer S F , Shahani U , Maas P , Weir A I , Rosenberg J R
Published: December 2014

1. Simultaneous recordings of cortical activity, recorded as the magnetoencephalogram (MEG), and the electromyogram (EMG) of the ipsilateral and contralateral first dorsal interosseous muscles (1DI) were made during maintained voluntary contractions. 2. The MEG recorded from a localized region of the sensorimotor cortex of the dominant hemisphere was coherent with the EMG from the contralateral 1DI muscle over a limited band of frequencies. The peak coherence was confined largely within the beta range of cortical activity (13‐35 Hz). Significant cortical activity at 10 Hz and 40‐50 Hz was not correlated with motor output. The MEG and EMG from the ipsilateral 1DI muscle were uncorrelated at all frequencies. 3. Significant coherence between the MEG and the EMG was associated with synchronous behaviour between the MEG and EMG in the time domain. 4. The results demonstrate that synchronized cortical activity contributing to MEG activity within the beta range of frequencies during maintained voluntary contractions is coupled to motor output at frequencies of motor‐unit activity associated with motor‐unit synchronization. This observation provides further evidence for the involvement of cortical neurones in the generation of motor‐unit synchronization. 5. We suggest that the coherence between MEG and contralateral EMG observed during maintained isometric contractions may provide an example of binding within the motor system.
Static and dynamic actions of cytoplasmic Ca2+ in the adaptation of responses to saturating flashes in salamander rods.
Authors: Matthews H R
Published: December 2014

1. In order to study the relative contribution to light adaptation of the various actions of Ca2+ in rod photoreceptors, changes in cytoplasmic calcium concentration ([Ca2+]i) were opposed by manipulating the calcium fluxes across the outer segment membrane at different times during the response to a bright flash. 2. When the outer segment was superfused with 0 Ca2+, 0 Mg2+,0 Na+ solution just before a bright flash, the period of response saturation was greatly prolonged. But if instead the solution change was made at progressively increasing times after the flash, the delay before the response recovered from saturation declined exponentially towards its value in Ringer solution with a time constant of around 1 s. In contrast, recovery time was little affected by stepping to 0 Ca+,0 Mg2+,0 Na+ solution before the flash and returning to Ringer solution shortly before the normal time of recovery from saturation. 3. When a bright flash was delivered just before the extinction of steady light, the response recovered from saturation progressively earlier as this steady intensity was increased. If, instead, the outer segment was transferred to 0 Ca2+,0 Mg2+,0 Na+ solution just before the bright flash then the time spent in saturation by the response was prolonged in darkness, but this additional delay progressively decreased as the steady intensity increased. 4. These results are consistent with the notion that the light‐induced reduction of the time spent in saturation by the bright flash response in Ringer solution resulted from the static decrease in [Ca2+]i induced by the background, while the additional delay in the recovery from saturation when further changes in [Ca2+]i were prevented stemmed from the abolition of the dynamic fall in [Ca2+]i during the flash response. 5. Analysis of the effects of steady light on the time spent in saturation by the bright flash response under these conditions suggests that actions of [Ca2+]i at, or soon after, the time of the flash are largely responsible for the graded changes which take place in the bright flash response during light adaptation, while rapid actions of [Ca2+]i at the time of response recovery also play a role in the adaptation of the steady response to background light itself. 6. These data have been interpreted in terms of differential actions of [Ca2+]i on ‘early’ stages (e.g. events leading to phosphodiesterase activation) and ‘late’ stages (e.g. guanylyl cyclase) in the transduction mechanism. A quantitative model is presented which suggests that actions of [Ca2+]i on ‘late’ stages play a proportinately larger role in background adaptation than actions on ‘early’ stages.
Intracellular acidification reduced gap junction coupling between immature rat neocortical pyramidal neurones.
Authors: Rörig B , Klausa G , Sutor B
Published: December 2014

1. Developmental changes in electrophysiological properties of pyramidal neurones correlated with the developmental decline in gap junction‐dependent dye coupling were investigated in coronal slices of rat prefrontal and sensorimotor cortex. Effects of intracellular acidification induced by application of weak organic acids on neuronal dye coupling, electrotonic parameters as well as synaptic potentials were examined using the patch clamp technique. Optical monitoring of intracellular pH revealed an acidic shift of 0.4‐0.5 pH units following sodium propionate application. 2. Dye coupling between layer II‐III neurones was prominent during the first two postnatal weeks. During this period, pre‐incubation of slices with 30 mM of the sodium salts of weak organic acids reduced the number of cells coupled to the injected neurones by 64%. 3. Between postnatal days 1 and 18, the mean neuronal input resistance decreased significantly (by 81.0%). Both the membrane time constant (tau 0) and the first equalizing time constant (tau 1) also showed a significant developmental decline of 25.8 and 65.8%, respectively. Electrotonic length decreased by 34.9%. The electrophysiological properties of neurones displayed a pronounced intercellular variability which decreased with on‐going development. 4. During the first two postnatal weeks, intracellular acidification led to a mean increase in neuronal input resistance of 55.9% and a mean decreae in electrotonic length of 22.2%. The membrane time constant was reduced by approximately 25% in the majority of neurones tested. Significant electrophysiological effects induced by intracellular acidification were not detected in uncoupled neurones from 18‐day‐old rats. 5. EPSP width at half‐maximal amplitude showed a substantial reduction of approximately 50%, while rise times of the non‐NMDA receptor‐mediated EPSP components displayed no significant change during development. Both weak organic acids, as well as the gap junction blocker 1‐octanol, reduced excitatory synaptic transmission independent of developmental age. 6. We conclude that gap junction permeability is regulated by intracellular pH in developing layer II‐III pyramidal cells in the rat neocortex. The prominent correlation between pH‐induced reduction in dye coupling and changes in electrophysiological cell properties suggests a significant influence of gap junctions on synaptic integration and information transfer in the immature neocortex.
Whole‐cell K+ currents in identified olfactory bulb output neurones of rats.
Authors: Wang X Y , McKenzie J S , Kemm R E
Published: December 2014

1. Voltage‐gated whole‐cell K+ currents have been investigated in olfactory bulb (OB) output (mitral/tufted) neurones from neonatal rats, which were retrogradely labelled by rhodamine or Fast Blue and identified after enzymatic dissociation. Forty‐five per cent of labelled neurones exhibited either phasic or non‐phasic spontaneous firing in cell‐attached configuration. 2. Four outward K+ currents have been identified in all such identified OB output neurones. They are the transient (IA), the delayed rectifier (IDK), and two Ca(2+)‐dependent (IK(Ca)) currents. No inward rectifier was detected. 3. The IA was activated at around ‐45 mV and reached its peak within 3‐10 ms. The decay phase could be described by single exponential distribution with the time constant of 45.2 +/‐ 3.8 ms at depolarizations 10‐60 mV from a holding potential of ‐70 mV. Its activation and steady‐state inactivation processes could be fitted with Boltzmann equations yielding half‐maximal activation potentials of 7.6 +/‐ 0.4 and ‐47.4 +/‐ 0.2 mV, respectively. It was sensitive to block by 4‐AP (1 mM) and by Zn2+ (1 mM). 4. The IDK was activated at potentials more positive than ‐30 mV, with half‐maximal activation at 21 mV. It was sustained during 1 s test pulses without apparent decay. It was blocked by TEA at a concentration of 20 mM. About 8% of the sustained current, in 11/24 cells tested, was found to resist block by a combination of all pharmacological agents tested. 5. Apamin at 100 nM blocked a TEA‐insensitive component which accounted for about 23% of the maximal sustained currents. Iberiotoxin (IbTX), which has been found to block maxi K+ currents more selectively than does charybdotoxin, reversibly blocked Ca(2+)‐activated K+ current, with a half‐maximal dose of about 100 nM in 8/13 OB output neurones tested. This accounted for 20% of the maximal sustained K+ current. The effect of IbTX was not observed in the presence of 20 mM external TEA. 6. Direct evidence is provided in this study regarding kinetic and pharmacological properties of four types of outward K+ channels in OB output neurones.
Activation by intracellular ATP of a potassium channel in neurones from rat basomedial hypothalamus.
Authors: Rowe I C , Treherne J M , Ashford M L
Published: December 2014

1. Cell‐attached recordings from isolated glucose‐sensitive hypothalamic neurones show that on removal of extracellular glucose there is an increased action current frequency concomitant with decreased single‐channel activity. Conversely activation of single K+ channels was observed when extracellular glucose was increased. Isolation of membrane patches into the inside‐out configuration following cell‐attached recording demonstrated the presence of an ATP‐activated K+ channel. 2. The ATP‐activated K+ channel was characterized by a mean single‐channel conductance of 132 pS in symmetrical 140 mM KCl solutions. Single‐channel open‐state probability (Po) was not calcium dependent, and the presence of calcium did not prevent activation of the channel by ATP. 3. Activation of the channel by ATP was concentration dependent and the Po of the ATP‐activated channel was unaffected by membrane voltage, regardless of the degree of activation elicited by ATP. 4. Open and closed time histograms were constructed from inside‐out and cell‐attached recordings and were consistent with a single open and two closed states. Channel openings were grouped in bursts. Application of ATP, in isolated patches, and glucose, in cell‐attached patches, increased the burst duration and number of bursts per second and decreased the slow closed‐state time constant. In neither case was there a significant change in the fast closed‐state time constant nor the open‐state time constant. 5. The non‐hydrolysable ATP analogue adenylylimidodiphosphate (AMP(PNP)) and ‘Mg2(+)‐free’ ATP produced little change in the Po of the ATP‐activated K+ channel when applied to the intracellular surface of excised patches. These results suggest that activation of this channel is via an enzymic mechanism. 6. ADP, GTP and GDP also activated the channel in a Mg(2+)‐dependent manner. ADP and ATP activated the channel in an additive manner and neither GTP nor GDP inhibited channel activity induced by ATP. 7. It is concluded that the ATP‐activated K+ channel observed in isolated inside‐out patches from hypothalamic neurones is the same as the channel activated by an increase in the concentration of extracellular glucose in cell‐attached recordings from glucose‐sensitive neurones.
Identification and properties of an ATP‐sensitive K+ current in rabbit sino‐atrial node pacemaker cells.
Authors: Han X , Light P E , Giles W R , French R J
Published: December 2014

1. Single myocytes were isolated from rabbit sino‐atrial (SA) node by enzymatic dissociation. Spontaneous pacemaker activity, whole‐cell and single‐channel currents were recorded under conditions known to modulate ATP‐sensitive K+ (KATP) channels. 2. The KATP channel openers, cromakalim and pinacidil, slowed or abolished the pacemaker activity, and caused hyperpolarization of the maximum diastolic potential (MDP). Glibenclamide, a KATP channel blocker, reversed these effects. Cromakalim‐ and pinacidil‐activated currents reversed near the potassium equilibrium potential, EK. Glibenclamide had no effect on the L‐type calcium current, ICa(L), the hyperpolarization‐activated inward current, If, or the delayed rectifier potassium current, IK. 3. Sodium cyanide, which inhibits mitochondrial ATP production, induced a macroscopic current that reversed near EK and was blocked by glibenclamide. 4. In excised, inside‐out patches from SA node cells, single KATP channels showed a slope conductance of 52 +/‐ 8 pS (mean +/‐ S.D.) when measurements were made at negative voltages in symmetric, 140 mM K+. Channels from ventricular myocytes showed a somewhat larger slope conductance (70 +/‐ 5 pS). 5. Raising the intracellular ATP concentration caused a concentration‐dependent reduction in the open probability of the KATP channels (IC50, 16 microM; Hill coefficient, approximately 1; at both pH 7.4 and 6.8). 6. In excised inside‐out patches, cromakalim or pinacidil induced significant increases in KATP channel activity in the presence of 50 microM or 1 mM intracellular ATP. This channel activity was blocked by glibenclamide. 7. Our results suggest that sino‐atrial node cells express a distinct isoform of KATP channel which may play an important role in pharmacological and pathophysiological modulation of pacemaker activity.
Patch‐clamp characterization of nicotinic receptors in a subpopulation of lamina X neurones in rat spinal cord slices.
Authors: Bordey A , Feltz P , Trouslard J
Published: December 2014

1. Nicotinic acetylcholine receptors (nAChRs) on lamina X neurones in neonate (P1‐P12) rat transverse thoracolumbar spinal cord slices were studied using the whole‐cell patch‐clamp technique. These visually selected neurones are located dorsal to the central canal, mainly in the ventral half of the dorsal commissure. 2. Pressure application of the nicotinic agonist 1,1‐dimethyl‐4‐phenyl‐piperazinium (DMPP) (1 mM) induced a rapid depolarization on which action potentials are superimposed. 3. At ‐50 mV, DMPP (1 mM), pressure ejected for 100 ms, induced a fast inward current with a mean amplitude of ‐280 pA (n = 28) in 90% of the neurones recorded. Superfusion of tetrodotoxin (TTX), a solution containing 0 Ca(2+)‐high Mg2+, CdCl2 or 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) did not abolish the DMPP‐induced current, which confirmed a direct postsynaptic effect of DMPP on recorded neurones. 3. The current‐voltage (I‐V) relationship for DMPP‐induced current exhibited a reversal potential of 0 mV (NaCl outside, potassium gluconate inside) and a strong inward rectification. 4. The DMPP‐induced responses were blocked by mecamylamine, hexamethonium and d‐tubocurarine (dTC) but were insensitive to alpha‐bungarotoxin and methyllycaconitine (MLA). 5. We conclude that lamina X neurones located dorsally to the central canal possess nicotinic acetylcholine receptors. Activation of these nicotinic receptors results in depolarization and generation of action potentials. These receptors may be involved in the modulation of the somato‐ and viscerosensory transmission.
The role of adenosine in functional hyperaemia in the coronary circulation of anaesthetized dogs.
Authors: Karim F , Goonewardene I P
Published: December 2014

1. The aim of this investigation was to determine the contribution of adenosine to coronary active hyperaemia in the dog denervated heart by using adenosine deaminase. 2. Beagles were anaesthetized with thiopentone sodium (500 mg, I.V.) and chloralose (100 mg kg‐1, LV.) and artificially ventilated. The hearts were denervate by bilateral cervical vagotomy and cardiac sympathectomy. Blood samples were collected from the coronary sinus via a cannula passed through the right external jugular vein. The anterior descending or circumflex branch of the left coronary artery was cannulated and perfused with blood from the left subclavian artery under systemic blood pressure through an electromagnetic flow probe and a perfusion circuit. The heart was paced (3 V, 0.2 ms and a suitable frequency) via two electrodes attached to the right atrium from 109 +/‐ 7.3 to 170 +/‐ 9.8 beats min‐4 (means +/‐ S.E.M.) for 3‐4 min, first during an infusion of the solvent, and then during an infusion of a solution of adenosine deaminase (5 U kg‐1 min‐1) into the circuit. 3. In seventeen tests in eight dogs, infusion of adenosine deaminase did not cause a significant change in the basal coronary blood flow nor in the immediate increase (within 10s) in blood flow induced by pacing the heart from its basal rate to 170 beats min‐1. However, adenosine deaminase did cause a significant attenuation by 58 +/‐ 5.2% (P < 0.05) of the increase in coronary blood flow induced at 3‐4 min of pacing from 31 +/‐ 4.6 to 43 +/‐ 5.8 ml min‐1 (100 g cardiac tissue)‐1. Concomitantly, the pacing‐induced increase in coronary vascular conductance (from 0.41 +/‐ 0.08 to 0.54 +/‐ 0.12 ml min‐1 (100 g)‐1 mmHg‐1) was reduced by 75 +/‐ 6.6% (P < 0.02) and the increase in myocardial O2 consumption (from 13 +/‐ 3.5 to 21 +/‐ 4.2 ml min‐1 (100 g)‐1) was reduced by 50 +/‐ 12% (P < 0.05) but without significant changes in oxygen extraction or myocardial contractility. 4. The results show that although adenosine is unlikely to play a significant role in the regulation of the basal coronary blood flow, it can play a major role in the coronary active (functional) hyperaemia induced by atrial pacing to a high rate in the denervated heart of anaesthetized dogs.
Ventilatory relief of the sensation of the urge to breathe in humans: are pulmonary receptors important?
Authors: Harty H R , Mummery C J , Adams L , Banzett R B , Wright I G , Banner N R , Yacoub M H , Guz A
Published: December 2014

1. The sensation of an urge to breathe (air hunger) associated with a fixed level of hypercapnia is reduced when ventilation increases. The aim of the present study was to investigate whether pulmonary receptors are important in this mechanism. 2. Five heart‐lung transplant (HLT) subjects and five control subjects were studied during periods of mechanical and spontaneous ventilation. End‐tidal Pco2 (PET,CO2) was increased by altering the level of inspired CO2. Throughout, subjects rated sensations of air hunger. Air hunger was also monitored during and immediately following maximal periods of breath‐holding. 3. When the level of mechanical ventilation was fixed, both groups experienced a high degree of air hunger when PET,CO2 was increased by about 10 mmHg. At similar levels of hypercapnia, both groups derived relief from approximately twofold increases in tidal volume, although relief was slightly less effective in HLT subjects. This was reversible, with decreases in the level of mechanical ventilation rapidly giving rise to increased ratings of air hunger. 4. With breath‐holding, all subjects obtained some respiratory relief within 2 s of the break point; there was no significant difference between the groups. 5. The results suggest that sensations of an urge to breathe induced by hypercapnia can be modulated by changes in tidal volume in the presumed absence of afferent information from the lung.
Facilitation of T‐type calcium current in bullfrog atrial cells: voltage‐dependent relief of a G protein inhibitory tone.
Authors: Alvarez J L , Rubio L S , Vassort G
Published: December 2014

1. The properties of the low‐threshold calcium current, ICa,T, were investigated in bullfrog isolated atrial cardiomyocytes using the whole‐cell, patch‐clamp technique under control conditions and during beta‐adrenergic stimulation. 2. The intracellular application of GTP gamma S or adenosine‐5'‐O‐3‐thiotriphosphate (ATP gamma S), poorly hydrolysable analogues of GTP and ATP, respectively, barely affected ICa,T amplitude in control conditions. beta‐Adrenergic stimulation effects were more marked in the presence of ATP gamma S. 3. The intracellular application of GDP beta S and ADP reduced ICa,T amplitude. In cells pretreated with pertussis toxin, ICa,T amplitude was significantly increased. In both conditions, the addition of isoprenaline was without effect. 4. Under both control and beta‐adrenergic‐stimulated conditions, a conditioning prepulse to +70 mV did not fully inactivate ICa,T; rather ICa,T facilitation often occurred after beta‐adrenergic stimulation. 5. In GTP gamma S‐ and ATP gamma S‐dialysed cells, ICa,T facilitation was generally observed after a prepulse; it was larger in the ATP gamma S dialysis. Facilitation was sustained but ended immediately upon cessation of conditioning prepulses. After beta‐adrenergic stimulation, facilitation was more marked in GTP gamma S‐ than in ATP gamma S‐dialysed cells. 6. ICa,T facilitation was prevented by the intracellular application of GDP beta S and by pertussis toxin pretreatment. 7. ICa,T facilitation developed markedly in the presence of intracellular cyclic AMP. This effect was prevented by pertussis toxin pretreatment of the cells. 8. It is thus proposed that ICa,T is under a double antagonistic control by both a Gs and a Gi protein. Furthermore, the double‐pulse‐induced facilitation of ICa,T results from a voltage‐dependent relief of the Gi protein inhibitory tone. Such an effect is increased by protein kinase A‐dependent phosphorylation, presumably of the Gi protein.
Inwardly rectifying K+ channels in freshly dissociated coronary endothelial cells from guinea‐pig heart.
Authors: von Beckerath N , Dittrich M , Klieber H G , Daut J
Published: December 2014

1. Inwardly rectifying K+ (IK(IR)) currents of freshly dissociated coronary endothelial cells from guinea‐pig heart were investigated with the perforated‐patch technique. 2. The whole‐cell current‐voltage relationship of endothelial cells showed strong inward rectification. Increasing the extracellular K+ resulted in an increase of inward currents. The slope conductance of the cells in the potential range negative to the calculated potassium equilibrium potential (EK) with 5, 60 and 150 mM external potassium was 0.18 +/‐ 0.14, 0.55 +/‐ 0.50 and 0.63 +/‐ 0.29 nS (mean +/‐ S.D.), respectively. 3. To quantify the steepness of inward rectification, the voltage dependence of the chord conductance of the cells was fitted with a Boltzmann function. The slope factor k describing the steepness of the relationship was 6.8 +/‐ 1.5 mV. 4. Extracellular barium induced a potential‐ and time‐dependent block of inward currents through endothelial KIR channels. Half‐maximum inhibition of IK(IR) currents was achieved with < or = 1 microM barium at a membrane potential of ‐70 mV in a solution containing 60 mM K+. 5. Whole‐cell inward currents revealed the opening and closing of single KIR channels. The single‐channel conductance was 26 +/‐ 3 pS with 60 mM external K+ and 33 +/‐ 6 pS with 150 mM external K+. 6. Our results suggest that the electrical properties of freshly dissociated endothelial cells are to a large extent determined by five to sixty active strong inwardly rectifying K+ (KIR) channels.
Differential oxygen sensitivity of calcium channels in rabbit smooth muscle cells of conduit and resistance pulmonary arteries.
Authors: Franco-Obregón A , López-Barneo J
Published: December 2014

1. Calcium currents were recorded from smooth muscle cells dispersed from conduit and resistance rabbit pulmonary arteries. We tested the hypothesis that Ca2+ channel activity was regulated by environmental O2 tension. 2. Conduit (proximal) and resistance (distal) myocytes differ in their Ca2+ channel density and responses to low PO2. Ca2+ current density in distal myocytes (20.7 +/‐ 7.4 pA pF‐1, n = 10) is almost twice the value in proximal myocytes (12.6 +/‐ 5.5 pA pF‐1, n = 39). In proximal myocytes, the predominant response to reductions in PO2 is inhibition of the calcium current (n = 12) at membrane potentials below 0 mV, whereas potentiation of current amplitude is observed in distal myocytes (n = 24). 3. Hypoxia also produces opposite shifts in the conductance‐voltage relationships along the voltage axis. The average displacements induced by low PO2 are +5.05 +/‐ 2.98 mV (n = 5) in proximal myocytes and ‐6.06 +/‐ 2.45 (n = 10) in distal myocytes. 4. These findings demonstrate longitudinal differences in Ca2+ channel density and O2 sensitivity in myocytes along the pulmonary arterial tree. These results may help to understand the differential reactivity to hypoxia of the pulmonary vasculature: vasodilatation in conduit arteries and vasoconstriction in resistance vessels.
Potentiation and depression of the M wave in human biceps brachii.
Authors: Cupido C M , Galea V , McComas A J
Published: December 2014

1. The effects of repeated excitation on the compound action potential, or M wave, of mammalian muscle fibres have been investigated in the human biceps brachii. 2. During continuous indirect stimulation at 10 and 20 Hz the mean voltage‐time area of the M wave doubled within the first minute, while the mean peak‐to‐peak amplitude increased by approximately half. The enlargement of the M wave was sustained during stimulation at 10 Hz but not at 20 Hz. Stimulation at 3 Hz caused a small increase which was significant for M wave amplitude only. 3. When the 20 Hz stimulation was performed under ischaemic conditions, the M wave first enlarged and then gradually declined. After 20 Hz stimulation was discontinued, the M wave increased in size; in the ischaemic experiments the release of the cuff produced a further, rapid augmentation. In both the ischaemic and non‐ischaemic experiments, the amplitudes and areas of the M waves during the recovery period became significantly larger than the resting values (range, 15‐60% at the endplate zone). 4. The mean muscle fibre impulse conduction velocity decreased to less than half the resting value during 20 Hz stimulation, with or without ischaemia, and then increased above the resting value during recovery. 5. On the basis of previous experiments in animals, the augmentation of the M wave was attributed to enhanced electrogenic Na(+)‐K+ pumping, and the biceps brachii appeared to be an excellent preparation for studying the time course of this enhancement.
Spike after‐depolarization and burst generation in adult rat hippocampal CA1 pyramidal cells.
Authors: Jensen M S , Azouz R , Yaari Y
Published: December 2014

1. Intracellular recordings in adult rat hippocampal slices were used to investigate the properties and origins of intrinsically generated bursts in the somata of CA1 pyramidal cells (PCs). The CA1 PCs were classified as either non‐bursters or bursters according to the firing patterns evoked by intrasomatically applied long ( > or = 100 ms) depolarizing current pulses. Non‐bursters generated stimulus‐graded trains of independent action potentials, whereas bursters generated clusters of three or more closely spaced spikes riding on a distinct depolarizing envelope. 2. In all PCs fast spike repolarization was incomplete and ended at a potential approximately 10 mV more positive than resting potential. Solitary spikes were followed by a distinct after‐depolarizing potential (ADP) lasting 20‐40 ms. The ADP in most non‐bursters declined monotonically to baseline (‘passive’ ADP), whereas in most bursters it remained steady or even re‐depolarized before declining to baseline (‘active’ ADP). 3. Active, but not passive, ADPs were associated with an apparent increase in input conductance. They were maximal in amplitude when the spike was evoked from resting potential and were reduced by mild depolarization or hyperpolarization (+/‐ 2 mV). 4. Evoked and spontaneous burst firing was sensitive to small changes in membrane potential. In most cases maximal bursts were generated at resting potential and were curtailed by small depolarizations or hyperpolarizations (+/‐ 5 mV). 5. Bursts comprising clusters of spikelets (‘d‐spikes’) were observed in 12% of the bursters. Some of the d‐spikes attained threshold for triggering full somatic spikes. Gradually hyperpolarizing these neurones blocked somatic spikes before blocking d‐spikes, suggesting that the latter are generated at more remote sites. 6. The data suggest that active ADPs and intrinsic bursts in the somata of adult CA1 PCs are generated by a slow, voltage‐gated inward current. Bursts arise in neurones in which this current is sufficiently large to generate suprathreshold ADPs, and thereby initiate a regenerative process of spike recruitment and slow depolarization.
Detection and modulation of acetylcholine release from neurites of rat basal forebrain cells in culture.
Authors: Allen T G , Brown D A
Published: December 2014

1. Nicotinic acetylcholine (ACh) receptor‐rich patches prepared from rat myotubes were used as focal ACh detectors to record the release of ACh from magnocellular basal forebrain (MBF) neurones from 11‐ to 14‐day‐old postnatal rats maintained in dissociated cell culture. 2. An action potential generated by intracellularly stimulating the MBF cell soma through a patch electrode induced a brief (mean tau(decay), 6.3 ms) short latency (1.35‐5.1 ms; median 3.1 ms) burst of nicotinic channel openings in the detector patch when the latter was positioned at discrete loci along the MBF neurites. Detected ACh concentrations ranged from approximately 480 nM to > 50 microM. Concentrations increased markedly during the first 14 days in vitro and were inversely related to response latency. 3. Sites of release were generally confined to the more proximal neurites within 100 microm of the cell body and were invariably associated with the presence of small (2‐3 microm diameter) phase‐dark puncta located at discrete intervals along the length of the neurites or at points where short collaterals branched from the main process. Release was never detected from the cell soma except under extreme non‐physiological conditions but could occasionally be elicited from growth cones at the ends of the shorter thicker neurites in the absence of a target cell. 4. Evoked release was abolished by tetrodotoxin (0.5 microM) and by superfusing with low Ca(2+)‐high Mg(2+)‐containing solutions (0.25 mM Ca(2+), 5 mM Mg(2+)). Myotube patch responses were antagonized by d‐tubocurarine (3 microM). 5. Muscarine (10 microM) inhibited release by 70 +/‐ 3% (n = 12 cells). This effect was antagonized by 100 nM methoctramine but not by 100 nM pirenzepine, indicating that it was mediated by M(2) muscarinic ACh receptors. 6. These results indicate that ACh release from the processes of magnocellular cholinergic basal forebrain neurones arises from highly specialized and discrete sites, and that it can be inhibited through activation of muscarinic receptors. It is suggested that the latter results from inhibition of presynaptic Ca(2+) channels and that it might be responsible for feedback autoinhibition of ACh release from cortical afferents of nucleus basalis neurones in vivo.
The hypothesis of the uniqueness of the oculomotor neural integrator: direct experimental evidence in the cat.
Authors: Godaux E , Cheron G
Published: December 2014

1. As far as horizontal eye movements are concerned, the well‐known hypothesis, not yet experimentally proved, of the common neural integrator states that the eye‐position signal is generated by a common network, regardless of the type of versional movement. The aim of this study was to evaluate the validity of this hypothesis by checking whether the sensitivity to eye position of the neurones of the nucleus prepositus hypoglossi (NPH) (the main component of the system integrating the different incoming velocity signals) would be the same regardless of the type of versional movement. 2. The discharge of sixty‐five NPH neurones was recorded in the alert cat during spontaneous eye movements made in the light and in response to sinusoidal rotations of the head in complete darkness. 3. For each NPH neurone, the sensitivity to eye position was determined from measurements carried out during intersaccadic fixation. The discharge rate of the studied neurone was plotted against eye position. The slope of the resulting regression line gave the sensitivity (measured during intersaccadic fixation in the light) of the neurone to eye position, which was termed K(f). 4. A new method was developed to measure the sensitivity to eye position (K(v)) of neurones during vestibular slow phases. The difficulty came from the fact that, during slow phases, eye velocity and eye position changed simultaneously and that each of those two variables could influence neuronal activity. For each neurone, the instantaneous firing rate was measured each time the eye passed through a given position during any slow phase generated during any vestibulo‐ocular reflex. At a given position, the discharge rate of the neurone under study was plotted against the eye velocity. From the resulting linear regression line, two interesting values were obtained: its slope, corresponding to the sensitivity of the neurone to eye velocity, R(v), (at that given eye position) and its ‘y'‐intercept, F(0), the interpolated firing rate when the eye velocity was zero. This procedure was repeated for different eye positions. The values of F(0) were then plotted against the eye positions. The slope of the resulting regression line gave the sensitivity (measured during vestibular stimulation) of the neurone to eye position, which was termed K(v). 5. The errors on the individual values of K(f) and K(v) were assessed in order to allow a statistical comparison at the single unit level. 6. We found that, for each of our sixty‐five neurones, the sensitivity to eye position measured during intersaccadic fixation in the light was equal to the sensitivity to eye position measured during the vestibulo‐ocular reflex (VOR) elicited in complete darkness. We conclude that our results favour the hypothesis of a unique horizontal oculomotor integrator for all versional movements.
Ca2+ current expression in pituitary melanotrophs of neonatal rats and its regulation by D2 dopamine receptors.
Authors: Gomora J C , Avila G , Cota G
Published: December 2014

1. We have examined the voltage‐dependent Ca2+ channel activity of rat melanotrophs during the early postnatal period. The cells were dissociated from pituitary intermediate lobes, kept in culture for 5‐24 h and then subjected to whole‐cell patch‐clamp experiments. 2. Like their adult counterparts, neonatal melanotrophs were able to generate Na+ currents, K+ currents and Ca2+ currents in response to membrane depolarization. Ca2+ currents were carried by both low‐ and high‐threshold Ca2+ channels. 3. High‐threshold Ca2+ current density decreased sharply between postnatal day 4 (P4) and P12. This period coincides with the onset of dopaminergic innervation within the intermediate lobe. Accordingly, the developmental decrease in Ca2+ current density was largely reversed by chronic in vivo treatment with sulpiride, a dopamine D2 receptor antagonist. 4. Prolonging the time in culture from 5 h to 8 days did not significantly alter the Ca2+ channel activity of P3 melanotrophs, whereas the high‐threshold Ca2+ current in previously innervated (P14) melanotrophs stayed small for the first 24 h and then increased 3‐fold during the subsequent 4‐5 days. This increase required RNA and protein synthesis and was prevented by adding D2 agonists to the culture medium. 5. These results provide evidence for a postnatal suppression of high‐threshold Ca2+ current expression in pituitary melanotrophs mediated by presynaptic dopamine neurons through D2 dopamine receptors.
Structural determinants of channel conductance in fetal and adult rat muscle acetylcholine receptors.
Authors: Herlitze S , Villarroel A , Witzemann V , Koenen M , Sakmann B
Published: December 2014

1. The structural basis of the developmentally regulated increase in endplate channel conductance in rat, where the gamma‐subunit of the fetal muscle acetylcholine receptor (gamma‐AChR) is replaced by the epsilon‐subunit in the adult muscle receptor (epsilon‐AChR), was investigated by analysing the structure of gamma‐ and epsilon‐subunit genes and by expressing recombinant AChR channels of different molecular composition in Xenopus oocytes and measuring their single‐channel conductance. 2. The gamma‐ and epsilon‐subunit genes each have twelve exons. In both subunits, the four homologous segments, designated M1, M2, M3 and M4, which are thought to contribute to the formation of the pore, are encoded by four separate exons, E7, E8, E9 and E12. 3. Chimaeric epsilon(gamma)‐ or gamma(epsilon)‐subunits were constructed from the parental epsilon‐ and gamma‐subunits, respectively. Exchanging the four hydrophobic segments (M1‐M4) of the gamma‐subunit for those of the epsilon‐subunit and vice versa completely reversed the difference in conductance between gamma‐AChR and epsilon‐AChR channels. 4. Effects of single‐ and multiple‐point mutations in M1‐M4 segments of gamma‐ and epsilon‐subunits indicate that the major determinants of the difference in conductance between fetal and adult endplate channels are located in the M2 segment. The key differences are the exchange of alanine/threonine (gamma‐subunit) for serine/isoleucine (epsilon‐subunit) in M2, and the lysine (gamma‐subunit) for glutamine (epsilon‐subunit) exchanges in the regions flanking the M2 segment.
Fusimotor and skeletomotor activities are increased with precision finger movement in man.
Authors: Kakuda N , Vallbo A B , Wessberg J
Published: December 2014

1. Impulses of eighteen muscle spindle afferents from finger extensor muscles were recorded from the radial nerve while subjects performed single joint finger movements of two kinds, i.e. routine and precision, which were nearly identical with regard to kinematics. 2. The firing rates of ten primary and two secondary spindle afferents were higher in the precision movements by more than 10%, although the difference reached statistical significance in only seven of them. In most cases when spindle firing was higher in precision movements the skeletomotor activity was higher as well. 3. The findings indicated that the fusimotor activity was often stronger with precision movements compared with routine movements. This result is in qualitative agreement with several studies on behaving cats, demonstrating higher fusimotor activity in more demanding motor tasks. On the other hand, the effects were much smaller in humans than in cats. Moreover, in contrast to findings from experiments in cats, no support was obtained for the hypothesis that fusimotor activity was adjusted independently of the skeletomotor activity in human finger muscles.
Postnatal development of the nociceptive withdrawal reflexes in the rat: a behavioural and electromyographic study.
Authors: Holmberg H , Schouenborg J
Published: December 2014

1. The postnatal development of nociceptive withdrawal reflexes was studied. In awake intact rats, forelimb, hindlimb and tail reflexes were recorded on videotape. In decerebrate spinal rats, electromyography (EMG) was used to record nociceptive withdrawal reflexes in musculi extensor digitorum longus (EDL), peronei, gastrocnemius‐soleus (G‐S) and biceps posterior‐semitendinosus (BP‐ST). Thermal (short‐lasting CO2 laser pulses) and mechanical stimulation were used. 2. In adults, nociceptive withdrawal reflexes were typically well directed and reflex pathways to single hindlimb muscles had functionally adapted receptive fields. By contrast, at postnatal day (P) 1‐7, the nociceptive withdrawal reflexes were often inappropriate, sometimes producing movements towards the stimulation, and EMG recordings revealed unadapted variable receptive fields. With increasing age, the nociceptive withdrawal reflexes progressively became well directed, thus producing localized withdrawal. Both withdrawal movements and spatial organization of the receptive fields were adult‐like at P20‐25. 3. Up to P25, reflex thresholds were more or less constant in both intact awake rats and spinal decerebrate rats, except in G‐S in which no nociceptive withdrawal reflexes were evoked from P20 on. After P25, mechanical, but not thermal, thresholds increased dramatically. 4. EMG recordings revealed that during the first three postnatal weeks, the latency of the CO2 laser‐evoked nociceptive withdrawal reflexes decreased significantly in peronei and BP‐ST, but not in EDL, and thereafter increased significantly in peronei, BP‐ST and EDL. The magnitude of the nociceptive withdrawal reflexes in these muscles increased markedly between P7 and P20 and showed little change thereafter. 5. Possible mechanisms underlying the postnatal tuning of the nociceptive withdrawal reflexes are discussed.
Interaction of L‐cysteine with a human excitatory amino acid transporter.
Authors: Zerangue N , Kavanaugh M P
Published: December 2014

1. The interaction of L‐cysteine with three excitatory amino acid transporter subtypes cloned from human brain (EAAT1‐3) was examined by measuring transporter‐mediated electrical currents and radiolabelled amino acid flux in voltage‐clamped Xenopus oocytes expressing the transporters. 2. L‐Cysteine was transported by the neuronal subtype EAAT3 (EAAC1) with an affinity constant of 190 microM and a maximal rate of flux similar to that of L‐glutamate; the relative efficacies (Vmax/K(m)) of the EAAT1 and EAAT2 subtypes for transporting L‐cysteine were 10‐ to 20‐fold lower. 3. Changing the ionization state of L‐cysteine by raising the external pH did not significantly change the apparent affinity, transport rate, or magnitude of currents induced by L‐cysteine, suggesting that both the neutral zwitterionic and anionic forms of the amino acid are transported with the same net charge stoichiometry. 4. In addition to competing with L‐glutamate for uptake by the neuronal carrier, L‐cysteine caused transporter‐mediated release of transmitter by heteroexchange; both actions would elevate extracellular glutamate concentrations and may thus contribute to the known excitotoxic actions of L‐cysteine in the brain. 5. Because the EAAT3 transporter is also expressed in tissues including kidney and intestine, the results suggest the possibility of a heretofore unrecognized mechanism of L‐cysteine uptake in peripheral tissues as well as in brain.
Activation of metabotropic glutamate receptor type 2/3 suppresses transmission at rat hippocampal mossy fibre synapses.
Authors: Kamiya H , Shinozaki H , Yamamoto C
Published: December 2014

1. The effects of metabotropic glutamate receptor (mGluR) agonists on excitatory transmission at mossy fibre‐CA3 synapses were studied in rat hippocampal slice preparations using both extracellular and whole‐cell clamp recording techniques. 2. Application of a novel and potent mGluR2/mGluR3‐specific agonist (2S,1'R,2'R,3'R)‐2‐(2,3‐dicarboxycyclopropyl)glycine (DCG‐IV, 0.1 microM) reversibly suppressed field excitatory postsynaptic potentials evoked by mossy fibre stimulation. DCG‐IV at the same concentration did not affect other glutamatergic excitatory transmissions at the commissural/associational input to CA3 or at the Schaffer collateral/commissural input to CA1 regions. 3. This suppressing effect of DCG‐IV on mossy fibre transmission was dose dependent and partly antagonized by a competitive mGluR antagonist (+)‐methyl‐4‐carboxylphenylglycine (1 mM). 4. The field potential changes induced by pressure application of glutamate (0.1 mM) to the stratum lucidum of the CA3 region was unaffected by 0.1 microM DCG‐IV. 5. In whole‐cell clamp experiments, 0.1 microM DCG‐IV suppressed excitatory postsynaptic currents evoked by mossy fibre stimulation without inducing detectable inward current in CA3 neurons, and paired‐pulse facilitation was enhanced by DCG‐IV application. 6. These results suggest that mGluR2/mGluR3 are specifically expressed at mossy fibre synapses in the hippocampal CA3 region, and activation of the receptor suppresses synaptic transmission by an action on a presynaptic site.