bluuebunny/crossref_metadata_2025 huggingface dataset:
Multimodal distribution of amplitudes of miniature and spontaneous EPSPs recorded in rat trigeminal motoneurones.
Authors: Min M Y , Appenteng K
Published: December 2014

1. The whole‐cell variant of the patch recording method has been used to obtain voltage recordings from trigeminal motoneurones in tissue slices (500 microns thick) taken from rats aged 8 days. Membrane properties (input resistance, membrane time constant and rheobase, i.e. threshold current required to elicit an action potential) of the motoneurones were determined and recordings made of the (untriggered) EPSP activity. 2. Untriggered EPSP activity was recorded in standard artificial cerebrospinal fluid (ACSF), ACSF with added tetrodotoxin (TTX) and in nominally Ca(2+)‐free ACSF with added TTX. In each case the amplitude distributions of single EPSPs were peaky and could be fitted by a model consisting of the sum of equidistant Gaussians (n = 7/9 cells). In contrast, the amplitude distribution of the noise was always unimodal. 3. All EPSP activity recorded in the presence of TTX was abolished by addition of 6‐cyano‐7‐nitroquinoxaline‐2‐3‐dione (CNQX; 10 microM), suggesting the activity was all mediated by glutamate acting primarily at AMPA/kainate receptors. 4. In the majority of cases, there was no correlation between the amplitude of EPSPs underlying each Gaussian and the EPSP rise time but there was a positive correlation between the EPSP half‐width and EPSP rise time. The rise times of EPSPs underlying the first, and all, fitted Gaussians were similar to that for the total sample of EPSPs in each motoneurone. Taken together, this suggests that the EPSPs underlying each Gaussian arise from inputs to different dendritic compartments, and that the range of compartments is similar for EPSPs underlying successive Gaussians. 5. Two conclusions are drawn. First, EPSPs of different dendritic origin have similar amplitudes at the soma. Second, the multimodal distribution of EPSP amplitudes recorded in the presence of TTX raises the possibility that individual boutons may contain multiple release sites, with each perhaps operating on a separate functional group of postsynaptic receptors.
Excitatory actions of GABA in developing rat hypothalamic neurones.
Authors: Chen G , Trombley P Q , van den Pol A N
Published: December 2014

1. Gramicidin‐perforated patch clamp recording was employed to study GABA‐mediated responses in rat hypothalamic neurones (n = 102) with an intracellular Cl‐ concentration unaltered by the pipette solution. 2. In young cultures after 1‐7 days in vitro (DIV), GABA induced depolarizing membrane potentials (+16.5 +/‐ 1.3 mV) that often surpassed the threshold for the firing of action potentials (‐42 +/‐ 1 mV) and resulted in an increase in neuronal activity. The depolarizing responses to GABA in young cultures were dose dependent. The concentration of GABA necessary to evoke the half‐maximal depolarization (EC50) was 2.8 microM. In contrast, GABA induced hyperpolarizing membrane potentials (‐12.0 +/‐ 1.4 mV) and a decrease in neuronal activity in older neurones (20‐33 DIV). Both the depolarization and the hyperpolarization induced by GABA were blocked by bicuculline, indicating a mediation by GABAA receptors. 3. The reversal potentials of the GABA‐evoked currents were between ‐40 to ‐50 mV during the first week of culture, and shifted to below ‐70 mV after 3 weeks of culture. In parallel, neurones that were dissociated from older animals (postnatal day 5) had a more negative reversal potential for the GABA‐evoked currents than cells from younger animals (embryonic day 15), suggesting that the negative shift of the reversal potential occurs both in vitro and in vivo. Our data suggest that the mechanism for GABA‐induced depolarization is the depolarized Cl‐ reversal potential found in young but not older neurones. 4. Consistent with the depolarizing response to exogenous application of GABA, some spontaneous depolarizing postsynaptic potentials in young cultures were insensitive to AP5‐CNQX, but were eliminated by bicuculline, indicating that synaptically released GABA mediated excitatory synaptic transmission in early development. 5. By combining a rapid computer‐controlled delivery of GABA with subthreshold positive current injections into recorded neurones, we found in young cultures that the GABA‐evoked depolarization could directly trigger action potentials, facilitate some depolarizing input to fire action potentials, and shunt other depolarizing input. Whether the GABA‐induced depolarization is excitatory or inhibitory would be determined by the reversal potential of the GABA‐evoked current, and the temporal relationship between GABA‐evoked depolarizations and other excitatory events. 6. We conclude that the reversal potential of the GABA‐evoked current shifts negatively from depolarizing to hyperpolarizing in developing hypothalamus. Consequently, GABA neurotransmission may serve both excitatory and inhibitory roles during early development.
Effects of FK506 and rapamycin on excitation‐contraction coupling in skeletal muscle fibres of the rat.
Authors: Lamb G D , Stephenson D G
Published: December 2014

1. The effects of the immunosuppressants FK506 and rapamycin were examined in mechanically skinned skeletal muscle fibres of rat in order to determine whether the FK506‐binding protein plays a role in the coupling between the voltage sensors and the Ca2+ release channels. 2. Both FK506 (1 microM) and rapamycin (1 microM) rapidly and reversibly potentiated Ca2+ release evoked by either depolarization of the transverse tubular system or caffeine application, suggesting a direct effect of the agents on the Ca2+ release channels. 3. In addition, repeated depolarizations in the presence of either FK506 (1 microM) or rapamycin (1 microM) caused irreversible loss of depolarization‐induced Ca2+ release, without preventing direct activation of the Ca2+ release channels by caffeine or low [Mg2+]. If a fibre was exposed to either immunosuppressant for a similar period (10 min) without stimulation, or if the voltage sensors were kept inactivated, there was little if any loss of coupling. 4. The loss of coupling was faster at higher drug concentrations, with 20 microM rapamycin causing 50% inhibition in 7‐8 min without stimulation; this was further accelerated by repeated depolarizations in the presence of the drug, but was not noticeably altered by direct activation of the release channels by repeated exposure to caffeine. The irreversible loss of coupling indicates that the FK506‐binding protein may play a vital role in enabling the voltage sensors to activate the Ca2+ release channels.
Cyclic GMP‐gated channels of bovine rod photoreceptors: affinity, density and stoichiometry of Ca(2+)‐calmodulin binding sites.
Authors: Bauer P J
Published: December 2014

1. Ca(2+)‐loaded vesicles of bovine rod outer segment (ROS) membranes were used to examine the influence of Ca(2+)‐calmodulin (Ca(2+)‐CaM) on the activity of cGMP‐gated channels. 2. In vesicles prepared from ROS membranes which were washed at zero free Ca2+, Ca(2+)‐CaM reduced the Ca2+ flux to maximally 40%. The dose‐response curve for activation of the cGMP‐gated channel had a half‐maximal value of 36.8 +/‐ 2 microM in the CaM‐free state, and of 55.6 +/‐ 5.2 microM in the Ca(2+)‐CaM‐bound state. In both cases the Hill coefficients were 2.2 +/‐ 0.2. 3. In vesicles prepared from ROS membranes which were washed at 100 microM Ca2+, the dose‐response curve was identical to the Ca(2+)‐CaM‐bound state. 4. Titration of the Ca(2+)‐CaM‐dependent decrease of the channel activity upon addition of 40 microM cGMP yielded half‐maximal Ca(2+)‐CaM concentrations (EC50CaM) which were linearly correlated with the concentration of membrane vesicles. Extrapolation of EC50CaM to infinite dilution of vesicles yielded a Ca(2+)‐CaM affinity constant for the cGMP‐gated channel of 1.01 +/‐ 0.20 nM. Hill analysis of the Ca(2+)‐CaM titrations resulted in a Hill coefficient of 1.36 +/‐ 0.15. 5. From the slope of the linear regression of EC50CaM plotted vs. the rhodopsin concentration, the molar ratio of rhodopsin to externally accessible Ca(2+)‐CaM binding sites of fused ROS membranes was determined to be 1439 +/‐ 109. Therefore, there are about 720 molecules of rhodopsin per Ca(2+)‐CaM binding site present in ROS. 6. Based on these data, a density of 560 Ca(2+)‐CaM binding sites micron‐2 is estimated for the plasma membrane of bovine ROS, suggesting that there are two Ca(2+)‐CaM binding sites per channel. 7. The Ca(2+)‐CaM effect did not become noticeable until the ROS membranes were hypotonically washed at free [Ca2+] below 100 nM, suggesting that an endogenous Ca(2+)‐binding protein was washed off in the absence of Ca2+. 8. If the endogenous Ca(2+)‐binding protein of bovine ROS membranes was washed off at zero Ca2+ and then Ca(2+)‐CaM added, Ca(2+)‐CaM could only be washed off again at free [Ca2+] below 100 nM. 9. These findings strongly suggest that the endogenous Ca(2+)‐binding protein of the bovine cGMP‐gated channel is CaM.
Voltage‐ and time‐dependent block of delayed rectifier K+ current in rabbit sino‐atrial node cells by external Ca2+ and Mg2+.
Authors: Ho W K , Earm Y E , Lee S H , Brown H F , Noble D
Published: December 2014

1. The properties of the delayed rectifier K+ current (IK) of rabbit isolated sino‐atrial node cells were investigated in high (140 mM) [K+]o using the whole‐cell‐clamp technique. 2. Hyperpolarizing clamp pulses from 0 mV induced an instantaneous current jump (I‐V relation linear) followed by a time‐dependent increase in inward current to a peak, whereas depolarizing clamp pulses induced little outward current. The peak I‐V relation showed a strong inward rectification. The inwardly rectifying current was blocked by E‐4031. 3. The inward K+ current induced by hyperpolarizing clamp pulses from 0 mV relaxed after reaching its peak. The rate of the relaxation increased as the membrane potential became more negative and concentrations of external Ca2+ or Mg2+ were increased. The steady‐state current was smaller as the relaxation of the current accelerated on increasing [Ca2+]o or [Mg2+]o. 4. Depolarizing clamp pulses from ‐80 mV induced an increase in inward current, reaching a steady state. The amplitude of the steady‐state current became smaller and the rate of current increase became slower as [Ca2+]o or [Mg2+]o was increased. 5. The effects of Ca2+ and Mg2+ are well explained by a time‐ and voltage‐dependent blockade of the K+ channel by these ions. The fractional electrical distance of the binding site calculated from the voltage dependence of the blocking rate constant is 0.69 for Ca2+ and 0.88 for Mg2+. The blocking rate constant at 0 mV for Ca2+ is about 15 times faster than that for Mg2+, indicating stronger effects of Ca2+. 6. A re‐interpretation of IK in sino‐atrial node cells is proposed: there are two independent gates (an activation gate which opens on hyperpolarization and an inactivation gate which closes on hyperpolarization) and a binding site for Ca2+ and Mg2+ inside the channel. Binding of these ions, which is facilitated by hyperpolarization, causes channel blockade, resulting in the observed voltage dependence of IK in physiological concentrations of Ca2+ and Mg2+.
Novel chloride‐dependent acid loader in the guinea‐pig ventricular myocyte: part of a dual acid‐loading mechanism.
Authors: Sun B , Leem C H , Vaughan-Jones R D
Published: December 2014

1. The fall of intracellular pH (pH1) following the reduction of extracellular pH (pH0) was investigated in guinea‐pig isolated ventricular myocytes using intracellular fluorescence measurements of carboxy‐SNARF‐1 (to monitor pH1). Cell superfusates were buffered either with a 5% CO2‐HCO3‐ system or were nominally CO2‐HCO3‐free. 2. Reduction of pH0 from 7.4 to 6.4 reversibly reduced pH1 by about 0.4 pH units, independent of the buffer system used. 3. In HCO3(‐)‐free conditions, acid loading in low pH0 was not dependent on Na(+)‐H+ exchange or on the presence of Na+. It was unaffected by high‐K+ solution, by voltage‐clamp depolarization, by various divalent cations (Zn2+, Cd2+, Ni2+ and Ba2+) and by the organic Ca2+ channel blocker diltiazem, thus ruling out proton influx through H(+)‐or Ca(2+)‐conductance channels or influx via a K(+)‐H+ exchanger. The fall also persisted in the presence of glycolytic inhibitors, or the lactate transport inhibitor, alpha‐cyano‐4‐hydroxy cinnamate. 4. In HCO3(‐)‐free conditions, acid loading in low pH0 was reversibly inhibited (by up to 85%) by Cl(‐)0 removal and was slowed by the stilbene drug DBDS (dibenzamidostilbene disulphonic acid). In contrast, the Cl(‐)‐HCO3‐exchange inhibitor DIDS (4,4'‐diisothiocyanatostilbene‐2,2'‐disulphonic acid) had no inhibitory effect. Acid loading is therefore mediated by a novel Cl(‐)‐dependent, acid influx pathway. 5. After switching to CO2‐HCO3(‐)‐buffered conditions, acid loading was doubled. It was still not inhibited by Na(+)‐free or high‐K+ solutions but was once again inhibited (by 78%) in Cl(‐)‐free solution. The HCO3(‐)‐stimulated fraction of acid loading was inhibited by DIDS. 6. We propose a model of acid loading in the cardiomyocyte which consists of two parallel carriers. One is Cl(‐)‐HCO3‐exchange, while we suggest the other to be a novel Cl(‐)‐OH‐exchanger (although we do not rule out the alternative configuration of H(+)‐Cl‐co‐influx). The proposed dual acid‐loading mechanism accounts for most of the sensitivity of pH1 to a fall of pH0.
A role for synaptic and network plasticity in controlling epileptiform activity in CA1 in the kainic acid‐lesioned rat hippocampus in vitro.
Authors: Bernard C , Wheal H V
Published: December 2014

1. Stimulation of the surviving afferents in the stratum radiatum of the CA1 area in kainic acid‐lesioned hippocampal slices produced graded epileptiform activity, part of which (> 20%) involved the activation of N‐methyl‐D‐aspartate (NMDA) receptors. There was also a failure of synaptic inhibition in this region. In this preparation, we have tested the effects of low‐frequency stimulation (LFS; 1 Hz for 15 min) on synaptic responses and epileptiform activity. 2. LFS resulted in long‐term depression (LTD) of excitatory synaptic potentials (EPSPs), long‐term decrease of population spike amplitudes (PSAs) and EPSP‐spike (E‐S) potentiation. Evoked epileptiform activity was reduced but neurons had a higher probability of discharge. LTD could be reversed by subsequent tetanic stimulation whereas E‐S dissociation remained unchanged. Synaptic and network responses could be saturated towards either potentiation or depression. However, E‐S potentiation was maximal following the first conditioning stimulus. 3. NMDA receptor‐mediated responses were pharmacologically isolated. LFS resulted in LTD of synaptic responses, long‐term decrease of PSAs and E‐S depression. These depressions could not be reversed by subsequent tetanic stimulation. alpha‐Amino‐3‐hydroxy‐5‐methylisoxazolepropionic acid (AMPA) and NMDA receptor‐mediated responses were then measured in isolation before and following conditioning stimuli. LFS was shown to simultaneously produce LTD of AMPA and NMDA receptor‐mediated responses. E‐S potentiation of the AMPA component and E‐S depression of the NMDA component occurred coincidentally. 4. LTD of AMPA and NMDA receptor‐mediated responses were shown to be NMDA dependent. In contrast, E‐S potentiation and depression occurred even when NMDA receptors were pharmacologically blocked. 5. These findings indicate that synaptic responses could be modified bidirectionally in the CA1 area of kainic acid‐lesioned rat hippocampus in an NMDA receptor‐dependent manner. However, E‐S dissociations were independent of the activation of NMDA receptors, hinting at mechanisms different from those of synaptic LTD. We suggest that changes in E‐S coupling were caused by a modification of the firing threshold of the CA1 pyramidal neurons. Furthermore, the firing mechanisms controlling NMDA and AMPA receptor‐mediated network activity appeared to be different. The possible use of LFS applied to the hippocampus as a clinical intervention to suppress epileptiform activity is discussed.
Bicarbonate secretion in interlobular ducts from guinea‐pig pancreas.
Authors: Ishiguro H , Steward M C , Wilson R W , Case R M
Published: December 2014

1. The transport of HCO3‐ across the luminal membrane of pancreatic duct cells was studied by monitoring the luminal pH of isolated guinea‐pig interlobular ducts after microinjection of an extracellular fluoroprobe, the dextran conjugate of 2'7'‐bis(2‐carboxyethyl)‐5(6)‐carboxyfluorescein (BCECF‐dextran). Luminal Cl‐ concentration was also measured by microfluorometry following microinjection of the dextran conjugates of 6‐methoxy‐N‐(4‐aminoalkyl)quinolinium bromide (ABQ‐dextran) and Cl‐NERF (Cl‐NERF‐dextran). 2. When HCO3‐/CO2 was admitted to the bath, a transient acidification of the duct lumen was observed, followed by a marked alkalinization. The latter was abolished when the luminal Cl‐ concentration was reduced to 25‐35 mM by replacement with glucuronate and may, therefore, be attributed to Cl(‐)‐HCO3‐ exchange at the luminal membrane. 3. Secretin, forskolin and acetylcholine stimulated HCO3‐ secretion into the lumen even when the luminal Cl‐ concentration was reduced to approximately 7 mM. Furthermore, agonist‐evoked HCO3‐ secretion was not inhibited by luminal glibenclamide, dihydro‐4,4'‐diisothiocyanostilbene‐2,2'‐disulphonic acid (H2DIDS) or 5‐nitro‐2‐(3‐phenylpropylamino)‐benzoic acid (NPPB). These observations are not easily reconciled with HCO3‐ transport across the luminal membrane being mediated by Cl(‐)‐HCO3‐ exchange in parallel with a Cl‐ conductance. 4. Agonist‐stimulated HCO3‐ secretion was blocked by omitting Na+ from the bath but not by addition of N‐methyl‐N‐isobutylamiloride (MIA) or bafilomycin A1. This supports our previous conclusion that HCO3‐ entry into duct cells from the extracellular fluid requires Na+ but is not dependent on Na(+)‐H+ exchange or vacuolar‐type H(+)‐ATPase activity. 5. The three actions of secretin on guinea‐pig pancreatic duct cells described in this and the accompanying paper ‐ stimulation of a relatively Cl(‐)‐insensitive luminal HCO3‐ efflux pathway, stimulation of basolateral Na(+)‐HCO3‐ cotransport, and lack of effect on intracellular pH‐ require the current model of pancreatic HCO3‐ secretion to be modified.
Activity‐dependent modulation of the presynaptic potassium current in the frog neuromuscular junction.
Authors: Miralles F , Solsona C
Published: December 2014

1. Changes in the electrical properties of frog motor nerve endings caused by the invasion of an action potential were studied by the perineural recording technique. Two equal supramaximal stimuli separated by a variable time interval were applied to the nerve trunk. The latency and amplitude of the deflections associated with the nodal Na+ current and presynaptic K+ current elicited by the second pulse were compared with control currents. 2. The deflection associated with the presynaptic K+ current elicited in response to the second stimulus was absent at the shortest interstimulus interval and showed a progressive increase in its amplitude as the interstimulus interval was lengthened, reaching values greater than control in most terminals. During the same period the nodal Na+ current did not change. 3. The experimental results were compared with a computer model of the distal axonal segment and its terminal. Response of the model to twin‐pulse stimulation was in marked disagreement with the experimental results unless an inactivating K+ channel, with properties derived ad hoc, was incorporated into the simulation. 4. These results suggest that in the first 6‐7 ms after a nerve impulse has invaded a frog motor nerve ending, maximal K+ conductance remains below the value at rest due to the fast inactivation of a K+ conductance. Following this, there is a period in which K+ conductance is greater than control values although the basis for this is unknown.
Membrane potentials of respiratory neurones during dizocilpine‐induced apneusis in adult cats.
Authors: Haji A , Pierrefiche O , Takeda R , Foutz A S , Champagnat J , Denavit-Saubié M
Published: December 2014

1. In the vagotomized cat, blockade of NMDA receptors by dizocilpine (MK‐801) produces an apneustic pattern of respiration characterized by a large increase in the duration of inspiration. 2. To identify dizocilpine‐induced disfacilitations and disinhibitions in respiratory neurones generating the respiratory rhythm, membrane potential and input resistance of augmenting inspiratory (I; n = 11) and post‐inspiratory (PI; n = 9) neurones were examined in the ventral respiratory group area, before and after administration of dizocilpine (0.1‐0.3 mg kg‐1 i.v.) in decerebrate, vagotomized, paralysed and artificially ventilated cats. 3. In I neurones, dizocilpine decreased the ramp depolarization and an 82% increase in input resistance was observed during inspiration. The inspiratory phase was prolonged, leading to a sustained level of depolarization during apneusis. The amplitude of stage 1 expiratory hyperpolarization decreased and its decay, which is normally slow, was faster. Throughout the remainder of expiration (stage 2) the membrane potential levelled off and the input resistance increased slightly (by 15%). 4. In PI neurones, dizocilpine depressed depolarization and suppressed firing in eight out of nine cells during the stage 1 expiratory phase. This was associated with a large (91%) increase of input resistance. The membrane potential switched quickly to stage 2 expiratory repolarization, during which a slight (19%) increase in input resistance occurred. 5. The hyperpolarization of PI neurones during early inspiration was reduced in amplitude by dizocilpine and input resistance was increased by 75% during inspiration, indicating that dizocilpine reduced the activity of the presynaptic inhibitory early‐inspiratory (eI) neurones. 6. We conclude that NMDA receptor blockade in the respiratory network disfacilitates eI, I and PI neurones during their active phase. Decreased inhibitory processes during the inspiratory phase probably play a major role in the prolongation of inspiration.
Binding sites contribute unequally to the gating of mouse nicotinic alpha D200N acetylcholine receptors.
Authors: Akk G , Sine S , Auerbach A
Published: December 2014

1. Single channel currents were recorded from HEK 293 cells expressing recombinant mouse adult (alpha 2 beta delta gamma) acetylcholine receptors (AChRs) containing a mutation at residue D200 of the alpha‐subunit. Rate and equilibrium constants for AChR activation were estimated from open and closed time obtained over a range of ACh concentrations. 2. Mutation of alpha D200 to asparagine (alpha D200N) dramatically slows the rate constant of channel opening, with adult AChRs slowing 100‐fold and embryonic AChRs slowing 400‐fold. the rate constant of channel closing increased 3‐fold, resulting in a decrease of the gating equilibrium constant of up to 1200‐fold. In contrast to channel gating steps, ACh‐binding steps are only modestly effected by alpha D200N. 3. Introduction of a potential glycosylation site in alpha D200N cannot account for the effect on channel gating because eliminating the consensus for glycosylation with the mutation alpha D200N + T202V fails to restore efficient gating. Gating is similarly impaired with the substitutions of E, K and Q at position alpha 200. 4. the agonists carbamylcholine and tetramethylammonium also activate the alpha D200N AChR, but with channel opening rates even slower than with ACh. The agonist dependence of the opening rate constant is similar in alpha D200N and wild type AChRs. 5. AChRs containing D200N at just one of the two alpha‐subunits show either small or large changes in the gating equilibrium constant, presumably due to the presence of the mutation at either the alpha delta or alpha epsilon/alpha gamma sites. The changes in free energy of channel gating show that the contribution of each binding site is nearly independent. However, the sites do not contribute equally to gating, as an alpha D200N mutation at the alpha epsilon or alpha gamma binding site slows channel opening relatively more than at the alpha delta site.
Retrograde signalling in depolarization‐induced suppression of inhibition in rat hippocampal CA1 cells.
Authors: Alger B E , Pitler T A , Wagner J J , Martin L A , Morishita W , Kirov S A , Lenz R A
Published: December 2014

1. We have investigated the phenomenon of ‘depolarization‐induced suppression of inhibition’ (DSI) using whole‐cell voltage‐clamp techniques in Ca1 pyramidal cells of rat hippocampal slices. DSI was induced by eliciting voltage‐dependent calcium (Ca2+) currents with 1 s voltage steps of +60 to +90 mV from the holding potential. DSI was apparent as a reduction in synaptic GABAA responses for a period of about 1 min following the voltage step. 2. TTX‐sensitive spontaneous IPSCs (sIPSCs) were susceptible to DSI, while TTX‐resistant miniature inhibitory postsynaptic current (mIPSCs) were not. Miniature IPSCs are ordinarily infrequent and independent of external Ca2+ in the CA1 region. To increase the frequency of mIPSCs and to induce a population of Ca(2+)‐sensitive mIPSCs, we increased the bath K+ concentration to 15 mM. The increased mIPSCs were also insensitive to DSI, however. 3. T whole‐cell pipette‐filling solution contained 5 mM 2(triethylamino‐N‐(2,6‐dimethyl‐phenyl)acetamide (QX‐314) to block voltage‐dependent Na+ currents and caesium to block K+ currents. Nevertheless, bath application of 50 microM 4‐aminopyridine (4‐AP) or 250 nM veratridine both clearly reduced DSI, evidently by acting at presynaptic sites. 4. The amplitudes of monosynaptically evoked IPSCs (elicited in the presence of 10 microM 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) and 50 microM 2‐amino‐5‐phosphonovaleric acid (APV)) were dramatically reduced during the DSI period. Weak stimulation produced small IPSCs and occasional ‘failures’ of transmission during the control period. The percentage of failures increased markedly during the DSI period. Moderate‐intensity stimulation produced larger IPSCs that were often composed of distinguishable multiquantal components. All‐or‐none failures of multiquantal IPSC components also occurred during DSI. 5. The degree of paired‐pulse IPSC depression did not change during DSI, whereas it was decreased, as expected, by baclofen. 6. We conclude that the data represent novel evidence that DSI is mediated by a retrograde signalling process possibly involving presynaptic axonal conduction block.
Properties of a novel K+ current that is active at resting potential in rabbit pulmonary artery smooth muscle cells.
Authors: Evans A M , Osipenko O N , Gurney A M
Published: December 2014

1. An outward current (IK(N)) was identified in rabbit pulmonary artery myocytes, which persisted after Ca(2+)‐activated and ATP‐sensitive K+ currents were blocked by TEA (10 mM) and glibenclamide (10 microM), respectively, and after A‐like (IK(A)) and delayed rectifer (IK(V)) K+ currents were inactivated by clamping the cell at 0 mV for 10 min. It was found in smooth muscle cells at all levels of the pulmonary arterial tree. 2. The relationship between the reversal potential of IK(N) and the extracellular K+ concentration ([K+]o) was close to that expected for a K(+)‐selective channel. Deviation from Nernstian behaviour at low [K+)o could be accounted for by the presence of an accompanying leakage current. 3. IK(N) is voltage gated. It has a low threshold for activation, between ‐80 and ‐65 mV, and activates slowly without delay. Activation follows an exponential time course with a time constant of 1.6 s at ‐60 mV. Deactivation is an order of magnitude faster than activation, with a time constant of 107 ms at ‐60 mV. 4. IK(N) showed a similar sensitivity to 4‐aminopyridine as IK(A) and IK(V), with 49% inhibition at 10 mM. The current was not blocked by microM quinine, which did inhibit IK(A) and IK(V), by 51 and 47%, respectively. 5. Activation of IK(N) was detected at potentials close to the resting membrane potential of pulmonary artery smooth muscle cells, under physiological conditions. Thus it is likely to contribute to the resting membrane potential of these cells.
Changes in maximal voluntary force of human adductor pollicis muscle during the menstrual cycle.
Authors: Phillips S K , Sanderson A G , Birch K , Bruce S A , Woledge R C
Published: December 2014

1. Muscle strength of the adductor pollicis (AP) was studied throughout the menstrual cycle to determine whether any variation in force is similar to the known cyclical changes in ovarian hormones. Three groups of young women were studied: trained regularly menstruating athletes (trained), untrained regularly menstruating (untrained) and trained oral contraceptive pill users (OCU). In addition a group of untrained young men was studied as controls. 2. Maximum voluntary force (MVF) of AP was measured over a maximum period of 6 months. Ovulation was detected by luteinizing hormone measurements or change in basal body temperature. There was a significant increase in MVF (about 10%) during the follicular phase of the menstrual cycle when oestrogen levels are rising, in both the trained and untrained groups. This was followed by a similar in MVF around the time of ovulation. Neither the OCU nor the male subjects showed cyclical changes in MVF.
Non‐averaged human brain potentials in somatic attention: the short‐latency cognition‐related P40 component.
Authors: Tomberg C , Desmedt J E
Published: December 2014

1. Non‐averaged scalp‐recorded brain potentials were studied in humans during selective attention to randomly intermixed series of stimuli to fingers. Physiological tests were use for validating the presence or absence of the short‐latency cognition‐related P40 electrogeneses in parietal cortex in the response to a single‐target stimulus (P40 signifies a positive polarity of about 40 ms peak latency). 2. To minimize interference from the electroencephalogram and noise we mapped single brain responses over the scalp and identified P40 topographies by an updated form of the numerical estimator Z for assessment of recorded potentials over time. We found that Z should exceed 0.96 for at least 15 ms for validation of the topographical congruity between the single P40 and an averaged P40 template. 3. Individual responses to 145 target finger stimuli correctly identified by the subject were analysed. P40 occurred only intermittently (34.5%) in a series of targets, but its voltage was unexpectedly large, exceeding the P40 voltage in averaged responses by a factor of about 10. 4. The usual assumption in the averaging method that the single brain responses combined in the average are stable but merely contaminated by unrelated noise was shown to be false for the cognition‐related P40, which was considerably underestimated because of its intermittency in the averaged single trials. 5. The reaction time of the subject was on average 19% shorter in the trials in which a P40 was present, thus suggesting that P40 can influence subsequent perceptual processing by the brain in the same trial. 6. The feasibility of identifying specific cognition‐related electrogeneses in single brain responses opens up the study of momentary shifts in brain processing strategies thereby allowing the neurophysiology of cognition to be based in real time.
Effects of barium, lanthanum and gadolinium on endogenous chloride and potassium currents in Xenopus oocytes.
Authors: Tokimasa T , North R A
Published: December 2014

1. The effects of multivalent cations on membrane currents recorded from Xenopus oocytes were studied. 2. The hyperpolarization‐activated chloride current was reversibly blocked by lanthanum; half‐maximal block occurred at a concentration of 8 microM. Zinc, cadmium, cobalt and nickel were less potent than lanthanum, and gadolinium, manganese, barium and strontium had no effect at a concentration of 100 microM. 3. The calcium‐activated chloride current was blocked by gadolinium (50 microM), and lanthanum, cadmium, cobalt, nickel and manganese were equally effective. The actions of gadolinium and lanthanum were almost irreversible, while partial (30‐80%) recovery was observed with the other cations. Zinc (100 microM) had no effect. 4. In lanthanum (100 microM), membrane depolarizations from ‐70 mV activated an outward potassium current that was partially blocked by barium (0.1‐2 mM). The barium‐sensitive current was confined to potentials less negative than ‐70 mV. The current consisted of a time‐independent as well as a time‐dependent component, the latter of which had voltage dependence similar to the M‐current. 5. It is proposed that lanthanum, gadolinium and barium can usefully separate these endogenous membrane currents in Xenopus oocytes.
Ca2+ sparks involving multiple Ca2+ release sites along Z‐lines in rat heart cells.
Authors: Parker I , Zang W J , Wier W G
Published: December 2014

1. High spatial resolution confocal imaging was used to investigate the fundamental nature of "Ca2+ sparks' in rat cardiac myocytes loaded with the fluorescent calcium indicator, fluo‐3. 2. The sites at which calcium sparks occurred (Ca2+ release sites) were packed closely and irregularly in transverse planes along Z‐lines (mean spacing between sites of 0.76 microns). In contrast, sites were spaced more regularly in the longitudinal direction, at intervals of 1.8 microns (i.e. the sarcomere length). 3. Diffusion of released Ca2+ was slower transversely (apparent diffusion coefficient, D, 7.9 microns 2 s‐1) than longitudinally (D, 17.1 microns 2 s‐1). 4. Frequently, discrete sites several hundred nanometres apart transversely activated in near synchrony. The probability of transverse synchronous activity fell to low levels (< 20%) at sites separated by more than 1.0 micron. Synchronous activation was not observed between sites on different Z‐lines (i.e. separated longitudinally by 1.8 microns). 5. High temporal resolution confocal microscopy (stationary spot) revealed Ca2+ sparks with "stepped' rises, consistent with multiple sites of origin. 6. We conclude that the Ca2+ spark as originally described is usually not an "elementary' event, in the sense of being indivisible, but is often comprised of yet smaller, triggered units of Ca2+ release.
The hyperpolarization‐activated current (Ih) and its contribution to pacemaker activity in rat CA1 hippocampal stratum oriens‐alveus interneurones.
Authors: Maccaferri G , McBain C J
Published: December 2014

1. The hyperpolarization‐activated current (Ih) and its role in pacemaking activity in rat hippocampal stratum oriens‐alveus interneurones was studied using whole‐cell and perforated patch‐clamp configurations. 2. Voltage‐clamp recordings revealed Ih as a slowly activating, inward current, activated by hyperpolarizing steps (holding potential, Vh = ‐40 mV), with a reversal potential close to ‐30 mV. Its activation curve ranged from approximately ‐50 to ‐120 mV with a mid‐activation point of ‐84.1 mV. 3. Ih was blocked by external application of Cs+ (2‐5 mM) and ZD7288 (100 microM), but not by Ba2+ (1 mM). 4. Ih was potentiated by both noradrenaline and isoprenaline by a mechanism consistent with a shift in the Ih activation curve. 5. Under current‐clamp conditions (Vh = ‐60 mV), ZD7288 induced a membrane hyperpolarization concomitant with an increase in the membrane input resistance and abolished the voltage sag generated by hyperpolarizing current injection. 6. Analysis of the current‐discharge relationship revealed that block of Ih differentially increased the firing frequency of spikes occurring early in the train compared with those occurring late in the discharge. 7. When applied to spontaneously firing cells, ZD7288 reduced the firing frequency by selectively altering the time course of the interspike interval, while minimally affecting other action potential characteristics. Similarly, isoprenaline increased the spontaneous firing frequency by an effect exclusively on the after‐hyperpolarization and interspike interval. 8. These results provide evidence for the involvement of Ih in the excitability and generation of spontaneous firing in hippocampal stratum oriens‐alveus interneurones.
On the dominant rhythm in the discharges of single postganglionic sympathetic neurones innervating the rat tail artery.
Authors: Johnson C D , Gilbey M P
Published: December 2014

1. In anaesthetized rats, using a focal recording technique, activity was recorded from single sympathetic postganglionic neurones innervating the caudal ventral artery of the tail. The following hypotheses were tested: (i) that the frequency of the dominant rhythmic discharge of the neurones can be different from the frequency of the central respiratory rhythm (as indicated by rhythmic phrenic discharge); and (ii) that the dominant sympathetic rhythm is not reliant on afferent feedback carried in aortic, sinus and vagus nerves. 2. Four types of preparation were used: spontaneously breathing (group 1), artificially ventilated (group 2), artificially ventilated with vagi cut (group 3), and artificially ventilated with vagus and sino‐aortic denervation (group 4). 3. The frequencies of the dominant sympathetic rhythm under control conditions were: group 1, 0.91 +/‐ 0.12 Hz (mean +/‐ S.E.M., n = 5); group 2, 0.81 +/‐ 0.04 Hz (n = 18); group 3, 0.83 +/‐ 0.03 Hz (n = 17); group 4, 0.95 +/‐ 0.06 Hz (n = 11). The frequency of the dominant sympathetic rhythm was different from that of the phrenic rhythm in thirty‐five out of fifty‐one cases. 4. The mean frequency of the dominant sympathetic rhythm was not influenced significantly by hypocapnic apnoea. 5. Hyperthermia increased the frequency of the phrenic rhythm whilst decreasing that of the dominant sympathetic rhythm. 6. In all cases the frequency of the dominant sympathetic rhythm was different from that of the artificial ventilation cycle. 7. It is concluded that the frequency of the dominant sympathetic rhythm can be different from that of central respiratory drive and that it is not "driven' by afferent feedback relayed via sinus, aortic and vagus nerves. 8. It is proposed that the dominant sympathetic rhythm is unlikely to be generated by a central respiratory oscillator.
The temporal profile of calcium transients in voltage clamped gastric myocytes from Bufo marinus.
Authors: McGeown J G , Drummond R M , McCarron J G , Fay F S
Published: December 2014

1. Decay in intracellular calcium concentration ([Ca2+]i) was recorded following step depolarizations in voltage clamped gastric myocytes from Bufo marinus. 2. Depolarizations (300 ms) to +10 mV were followed by three phases of [Ca2+]i decay with repolarization to both ‐110 and ‐50 mV. The decline was initially rapid (mean fractional decay rate = 81 +/‐ 11%s‐1 at ‐110 mV), then slowed (decay rate = 14 +/‐ 2%s‐1) and finally accelerated again (decay rate = 24 +/‐ 3%s‐1; n = 19). 3. The initial phase of rapid decay became shorter as the length of the depolarizing pulse increased but was unaffected by changes in pulse voltage. 4. The delayed acceleration in [Ca2+]i decay was no longer seen when the duration of the depolarizing pulses was reduced to 100 ms, but was clearly evident following 500 ms pulses. This phase was abolished when the depolarizing voltage was altered to minimize the rise in [Ca2+]i. 5. Ryanodine and caffeine had no effect on the temporal profile of [Ca2+]i decay. 6. Removal of extracellular Na+ decreased the decay rate during all three phases at ‐110 mV, but this effect was particularly marked for the initial rapid phase of decay, the rate of which was reduced by 75%. A delayed increase in decay rate was still seen. 7. Inhibition of mitochondrial Ca2+ uptake with cyanide, carbonyl cyanide p‐trifluoromethoxy‐phenylhydrazone or Ruthenium Red had no effect on the initial rate of [Ca2+]i decay but blocked the delayed acceleration. 8. These results are discussed in terms of a model in which rapid influx of Ca2+ produces a high subsarcolemmal [Ca2+], favouring rapid Ca2+ removal by near‐membrane mechanisms, particularly Na(+)‐Ca2+ exchange. Mitochondrial Ca2+ removal produces a delayed increase in [Ca2+]i decay if the global [Ca2+]i is raised high enough for long enough.
Ryanodine‐sensitive calcium stores involved in neurotransmitter release from sympathetic nerve terminals of the guinea‐pig.
Authors: Smith A B , Cunnane T C
Published: December 2014

1. Intracellular and focal extracellular recording techniques were used to study neurotransmitter release mechanisms in postganglionic sympathetic nerve terminals in the guinea‐pig isolated vas deferens. 2. High concentrations of the selective N‐type calcium channel blocker omega‐conotoxin GVIA abolished the release of the neurotransmitter ATP evoked by trains of low‐frequency stimuli. However, in the presence of high concentrations of the blocker, a ‘residual release’ persisted at higher frequencies. 3. Residual release was dependent on calcium entry through a pharmacologically distinct voltage‐dependent calcium channel. 4. Residual release was inhibited by ryanodine in a use‐ and time‐dependent manner and this inhibitory effect was potentiated by caffeine. The inhibitory effect of ryanodine on residual release was reversed by 4‐aminopyridine. 5. These findings indicate that calcium‐induced calcium released from intraneuronal stores plays an important role in action potential‐evoked neurotransmitter release mechanisms in postganglionic sympathetic nerve terminals.
Cross‐bridge detachment and attachment following a step stretch imposed on active single frog muscle fibres.
Authors: Piazzesi G , Linari M , Reconditi M , Vanzi F , Lombardi V
Published: December 2014

1. The time course of cross‐bridge detachment‐attachment following a step stretch was determined in single frog muscle fibres (at 4 degrees (1 and 2.1 microns sarcomere length) by imposing, under sarcomere length control by a striation follower, test step releases of various amplitudes (2‐13 nm per half‐sarcomere) at successive times (4‐55 ms) after a conditioning stretch of approximately 4 nm per half‐sarcomere. 2. The comparison with the control tension transients, elicited by releases not preceded by the conditioning stretch, shows that, early after the conditioning stretch, the quick tension recovery following small releases is depressed and the quick tension recovery following large releases is potentiated. Both effects are expected as a consequence of the strain produced in the cross‐bridges by the conditioning stretch. 3. These effects disappear and the tension transient is reprimed, indicating substitution of freshly attached cross‐bridges for strained cross‐bridges, with a time constant of approximately 10 ms. 4. A novel multiple‐exponential equation, based on the hypothesis of complete substitution of freshly attached cross‐bridges for the cross‐bridges that underwent the stretch, has been used to fit the whole tension transient following step stretches of different sizes (2‐6 nm per half‐sarcomere). For a stretch of 4 nm, the time constant of the exponential process responsible for cross‐bridge detachment (tau d, 9.3 ms) almost coincides with the time constant of repriming as measured by the double‐step experiments. The time constant of the exponential process representing the cumulative effects of attachment and force generation (tau 3) is 13.6 ms. 5. For stretches of different sizes the amount of quick tension recovery attributable to the reversal of the working stroke elicited by the stretches is estimated by subtracting, from the original tension transient, the contribution to tension recovery due to detachment‐attachment of cross‐bridges as estimated by the multiple‐exponential analysis. Following this calculation, the structural change in the myosin heads responsible for the reversal of the working stroke can be 2 nm at maximum, suggesting that the elastic component in the cross‐bridges is at least twice as rigid as previously thought.
Modulation of Na+,K(+)‐ATPase activity by a tyrosine phosphorylation process in rat proximal convoluted tubule.
Authors: Féraille E , Carranza M L , Rousselot M , Favre H
Published: December 2014

1. In the rat kidney proximal convoluted tubule, epidermal growth factor and insulin have been reported to stimulate Na+ reabsorption. Because most of the effects of these growth factors are mediated by a process of tyrosine phosphorylation and Na+,K(+)‐ATPase drives Na+ reabsorption, the influence of tyrosine kinases and tyrosine phosphatases on Na+,K(+)‐ATPase activity located in the proximal convoluted tubule was evaluated. 2. Activation of receptor tyrosine kinases by epidermal growth factor and insulin stimulated ouabain‐sensitive 86Rb+ uptake. The effects of epidermal growth factor and insulin were prevented by genistein, a tyrosine kinase inhibitor, but were unaffected by GF109203X, a protein kinase C inhibitor. 3. Inhibition of tyrosine phosphatases by orthovanadate (10(‐7) and 10(‐6)M) mimicked the effects of activation of receptor tyrosine kinases: stimulation of the ouabain‐sensitive 86Rb+ uptake and of the hydrolytic activity of Na+,K(+)‐ATPase under rate‐limiting Na+ concentration, and absence of modification of the maximal activity (Vmax) of the enzyme. The effects of orthovanadate and insulin on the ouabain‐sensitive 86Rb+ uptake were not additive. 4. The present results show that both activation of receptor tyrosine kinases and inhibition of tyrosine phosphatases stimulate the Na+,K(+)‐ATPase activity through a common mechanism. Thus, a tyrosine phosphorylation process directly controls the Na+,K(+)‐ATPase activity and contributes to the physiological control of water and solute reabsorption in the proximal convoluted tubule.
Delayed autoregulation of the Ca2+ signals resulting from capacitative Ca2+ entry in bovine pulmonary artery endothelial cells.
Authors: Madge L , Marshall I C , Taylor C W
Published: December 2014

1. In calf pulmonary artery endothelial (CPAE) cells loaded with fura‐2, the effects of ATP on Ca2+ entry were mediated entirely by the ability of P2U purinoceptors to stimulate InsP3 formation, empty intracellular Ca2+ stores and thereby activate capacitative Ca2+ entry. 2. Restoration of extracellular Ca2+ to cells with empty intracellular stores evoked transient increases in cytosolic [Ca2+] ([Ca2+]i) which then declined to an elevated plateau. These overshoots in [Ca2+]i were not a consequence of store refilling nor of desensitization of the capacitative pathway. Similar responses were recorded from cells in which Ca2+ uptake into mitochondria had been inhibited by microinjection of Ruthenium Red. The amplitudes of the capacitative Ca2+ signals decreased at lower extracellular [Ca2+], but [Ca2+]i invariably overshot before slowly declining to an elevated plateau. Even modest increases in [Ca2+]i therefore caused a delayed attenuation of the Ca2+ signal evoked by capacitative Ca2+ entry. 3. Modest pre‐elevation of [Ca2+]i inhibited the ability of subsequent capacitative Ca2+ entry to further increase [Ca2+]i. The onset of the inhibition was slow (half‐time (t1/2), approximately 100 s) and more tightly correlated with the preceding peak [Ca2+]i than with the [Ca2+]i immediately preceding Ca2+ entry. Recovery was also slow and complete only after [Ca2+]i had returned to its basal level for 320 +/‐ 3 s. 4. In thapsigargin‐treated cells loaded with mag‐fura‐2, the peak [Ca2+]i that followed restoration of extracellular Ca2+ was accompanied by an abrupt approximately 2.5‐fold decrease in the rate of Mn2+ entry, which then continued indefinitely at the reduced rate, demonstrating a rapid partial inactivation of the capacitative pathway. 5. The half‐time for Ca2+ removal from the cytosol was significantly slower during the rising (t 1/2 = 22 +/‐ 2.5 s) than during the falling (t 1/2 = 7.1 +/‐ 0.7 s) phase of the Ca2+ overshoot evoked by addition of extracellular Ca2+ to thapsigargin‐treated cells. 6. We conclude that an increase in [Ca2+]i rapidly inhibits the capacitative pathway and more slowly activates mechanisms that remove Ca2+ from the cytosol. Reversal of either or both of these regulatory mechanisms can occur only a considerable time after [Ca2+]i has been completely restored to its resting level. These mechanisms are likely to protect cells from excessive increases in [Ca2+]i and contribute to oscillatory changes in [Ca2+]i.