bluuebunny/crossref_metadata_2025 huggingface dataset:
Properties of ionic currents induced by external ATP in a mouse mesodermal stem cell line.
Authors: Kubo Y
Published: December 2014

1. ATP was puff applied to cells of a mesodermal stem cell line, C3H10T1/2, and the responses were studied by whole‐cell patch clamp recording. 2. In 91% of the cells (90/99), K+ current lasting for tens of seconds was observed after several seconds latency. The current showed outward rectification. In 10% of the cells (9/99), ATP induced Cl‐ current which also lasted for tens of seconds after several seconds latency, but showed little rectification. In 6% of these cells (5/99), both K+ and Cl‐ currents were induced by ATP. 3. The K+ current induced by ATP was dose dependent, with a Kd of 0.4 microM. The effects of ATP analogues were tested at a concentration of 20 microM. ADP and ATP‐gamma‐S induced the K+ current, while AMP and adenosine did not. alpha, beta‐Methylene ATP produced a diminished K+ current. 4. The ATP‐induced K+ current was not observed when EGTA in the internal solution was raised from 0.1 to 5 mM. In Fluo‐3‐loaded cells, an increase in intracellular Ca2+ concentration induced by the application of ATP was observed, and the time course was similar to the induced K+ current. Both the increase in intracellular Ca2+ and the K+ current were induced by ATP even in Ca(2+)‐free external solution. Ryanodine (50 microM) in the external solution did not affect the ATP response, and application of 10 mM‐caffeine alone to the external solution did not induce any response. 5. The variance of the steady‐state fluctuations in the course of the ATP‐induced slow K+ current was analysed. The single‐channel conductance was estimated as 2.7 pS at 0 mV with external and internal K+ concentrations of 5 and 140 mM respectively. The K+ current was not affected by apamin at concentrations of up to 1 microM but was reduced to one‐third by 140 mM‐tetraethylammonium (TEA). 6. It was concluded that puff‐applied ATP has two main effects in the mesodermal stem cells: an increase in the intracellular Ca2+ concentration and a succeeding hyperpolarization due to the Ca(2+)‐activated K+ conductance which is present in this cell. The significance of the increase in intracellular Ca2+ caused by ATP is discussed.
Effect on ventilation of papaverine administered to the brain stem of the anaesthetized cat.
Authors: Berkenbosch A , Olievier C N , DeGoede J , Kruyt E W
Published: December 2014

1. To investigate whether cerebral vasodilatation by itself contributes to the decrease in ventilation as found during brain stem hypoxia the role of cerebral vasodilatation on minute ventilation was investigated in twelve cats anaesthetized with alpha‐chloralose‐urethane. 2. Cerebral vasodilatation in the medulla oblongata was produced by adding papaverine to the blood perfusing the brain stem. 3. Papaverine at concentrations of 10‐35 micrograms per millilitre of blood had an appreciable depressant effect on ventilation. At a concentration of 14.3 micrograms ml‐1 the depression in ventilation averaged 0.7 +/‐ 0.1 l min‐1. 4. The ventilatory response to stepwise changes in papaverine concentration could be adequately described with a single exponential function with a time delay. 5. The time constant of the ventilatory response following a step increase in papaverine concentration (134 +/‐ 15 s) was longer than that of the step decrease (105 +/‐ 10 s) in concentration (P = 0.034). The time delays of the ventilatory response (88 +/‐ 21 s and 53 +/‐ 8 s respectively) were not significantly different (P = 0.126). 6. The ventilatory response to stimulation of the peripheral chemoreceptors by hypoxia and of the central chemoreceptors by hypercapnia was not impaired by papaverine. 7. The results support the hypothesis that cerebral vasodilatation by itself contributes to the decrease in ventilation by brain stem hypoxia.
Development of GABA‐mediated, chloride‐dependent inhibition in CA1 pyramidal neurones of immature rat hippocampal slices.
Authors: Zhang L , Spigelman I , Carlen P L
Published: December 2014

1. gamma‐Aminobutyric acid (GABA)‐mediated, Cl(‐)‐dependent inhibitory postsynaptic potentials (IPSPs) and GABA currents in immature rat hippocampal CA1 neurones were studied using the whole‐cell recording technique in brain slices. 2. IPSPs evoked by electrical stimulation were observed in postnatal 2‐ to 5‐ (PN2‐5), 8‐ to 13‐(PN8‐13) and 15‐ to 20‐(PN15‐20)day‐old CA1 neurones. In the presence of glutamate receptor blockers 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) and D‐2‐amino‐5‐phosphonovaleric acid (APV), the reversal potential for the IPSP (EIPSP) was near the resting membrane potential (RMP) in the PN2‐5 neurones, but 13 and 25 mV more negative than the RMP in PN8‐13 and PN15‐20 neurones respectively. IPSPs and GABA currents were blocked by the GABAA‐receptor antagonists bicuculline or picrotoxin. 3. The reversal potential for somatic GABA currents (EGABA) was examined in the presence of tetrodotoxin (TTX). There was a strong dependence of the EGABA upon the patch pipette [Cl‐] ([Cl‐]p). indicating that the GABA currents were mediated by a Cl‐ conductance. In PN2‐5 neurones, EGABA agreed with the value predicted by the Goldman‐Hodgkin‐Katz equation at given concentrations of internal and external anions permeable through GABA‐activated Cl‐ channels, whereas EGABA in older neurones was 8‐18 mV more negative. 4. Examination of the relations between EGABA, holding potential, [Cl‐]p and resting conductance indicated that the membrane of the PN2‐5 neurones was readily permeable to Cl‐ which followed a passive Donnan equilibrium. Passive distribution of Cl‐ played a decreasing role in PN8‐13 neurones and in PN15‐20 neurones. 5. To assess the contribution of outward Cl‐ co‐transport, bath applications of high K+ or furosemide were performed. High K+ and furosemide caused a reversible positive shift of EGABA in PN15‐20 neurones. Raising the temperature moved EGABA to a more negative potential, with a Q10 of 5 mV. A similar change of EGABA in response to high K+, but not to furosemide, was found in PN8‐13 neurones. 6. The present data indicate the existence of GABAA‐mediated inhibitory synaptic connections in CA1 neurones at the earliest stages of postnatal life. During the first postnatal week, Cl‐ ions are passively distributed and the EIPSP and EGABA are near the RMP.(ABSTRACT TRUNCATED AT 400 WORDS)
Properties of periodontal mechanoreceptors supplying the cat's lower canine at short and long periods after reinnervation.
Authors: Loescher A R , Robinson P P
Published: December 2014

1. The properties of periodontal mechanoreceptors innervating the lower canine teeth of cats have been determined 6 weeks and 1 year after sectioning the inferior alveolar nerve. Recordings were made from single fibres dissected from the nerve central to the injury site, whilst forces were applied at right angles to the long axis of the tooth. 2. The range of directions over which each mechanosensitive unit responded (are of sensitivity) was established. Forces were then applied in the direction of maximum sensitivity and the maximum discharge frequency, dynamic index, adaptation rate, and the force threshold at three rates of force application (0.25, 2 and 20 N s‐1) were determined. 3. Data from 115 units characterized 6 weeks after nerve section revealed a significantly reduced mean dynamic index and raised mean force threshold to forces applied at 2 and 0.25 N s‐1, when compared with the controls. These units were, however, more sensitive than those examined 12 weeks after nerve section in a previous study (Loescher & Robinson, 1989b). 4. Data from 158 units characterized 1 year after nerve section revealed a significantly narrower mean are of sensitivity, reduced mean maximum discharge frequency, lower mean dynamic index and raised mean threshold to forces applied at 0.25 N s‐1, when compared with the controls. 5. Bone overlying the roots of the reinnervated teeth was removed in order to localize receptors in the underlying periodontal ligament. Forty‐six units were localized 6 weeks after nerve section and thirty‐eight 1 year after nerve section. The mean conduction velocity of these units was significantly reduced both 6 weeks and 1 year after nerve section compared to the controls. One unit was found which branched to innervate receptors at two locations within the ligament, and one unit was found which branched to innervate a receptor in the ligament and another in the overlying skin. 6. The higher level of sensitivity of receptors when first reinnervated than at later stages is attributed to their immaturity. This immaturity may also contribute to the paraesthesia which is experienced by patients at a corresponding stage in recovery from nerve injury. The persistent reduction in sensitivity of units 1 year after nerve injury suggests that changes occur which have a permanent effect on their properties.
Crossed reflex actions from group II muscle afferents in the lumbar spinal cord of the anaesthetized cat.
Authors: Arya T , Bajwa S , Edgley S A
Published: December 2014

1. The effects of electrical activation of muscle nerves at strengths sufficient to activate group II afferents in contralateral hindlimb motoneurones have been investigated by intracellular recording from motoneurones in alpha‐chloralose‐anaesthetized cats in which the spinal cord was intact or transected at a low thoracic level. 2. A large proportion of the motoneurones encountered could be influenced by activation of contralateral group II afferents. With the spinal cord intact, the predominant effect of contralateral group II afferents was inhibitory in almost all motoneurones, including extensors. After spinal transection the same stimuli elicited EPSPs in extensor motoneurones and IPSPs in flexors. 3. Both with the spinal cord intact and following spinal transection, group II afferents from particular nerves had powerful effects, whereas others were without effect in most neurones and had weak effects in others. Stimulation of gastrocnemius‐soleus or of the hamstring nerves was largely ineffective, evoking weak effects in a small minority of motoneurones. Stimulation of the quadriceps, tibialis anterior‐extensor digitorum longus and flexor digitorum longus nerves had powerful effects and in a high proportion of motoneurones. 4. The minimal latencies of the IPSPs evoked prior to spinalization were directly comparable to those of IPSPs evoked from ipsilateral group II afferents. The possibility that the mid‐lumbar region, where many neurones which mediate ipsilateral group II reflexes are located, might mediate these crossed effects is discussed.
Calcium current and charge movement of mammalian muscle: action of amyotrophic lateral sclerosis immunoglobulins.
Authors: Delbono O , García J , Appel S H , Stefani E
Published: December 2014

1. The Vaseline‐gap voltage clamp technique was used to record dihydropyridine (DHP)‐sensitive Ca2+ currents (ICa) and charge movement in single cut fibres from the rat extensor digitorum longus (EDL) muscle. Amyotrophic lateral sclerosis (ALS) immunoglobulin G (IgG) action on ICa and charge movement has been characterized. 2. ALS IgG reduced ICa amplitude. The peak ICa of EDL fibres (mean +/‐ S.E.M.) at 0 mV, expressed as amperes per membrane capacitance, was ‐4.79 +/‐ 0.029 A F‐1, while after 30 min incubation in ALS IgG it was ‐2.52 +/‐ 0.04 A F‐1. IgG from healthy patients, and from patients with other diseases (familial ALS, myasthenia gravis, chronic relapsing inflammatory polyneuritis, multiple sclerosis and one sample from Lambert‐Eaton syndrome, LES) did not affect ICa, while IgG from patients with Guillain‐Barré syndrome and one other sample from a patient with LES affected the ICa in a similar way as ALS IgG. 3. The time constant of ICa activation (alpha m) at 0 mV was 44.8 +/‐ 1.4 ms in control, and 36.6 +/‐ 1.5 ms after an incubation of 30 min in ALS IgG. The steady‐state activation curve (m infinity) was shifted to more positive potentials by ALS IgG. 4. The rate constants of activation (range ‐20 to 30 mV) were altered by ALS IgG: alpha m decreased while beta m increased. These data suggest that ALS IgG favours the permanence of the Ca2+ channels in the closed state. 5. The time constant of Ca2+ channels deactivation at ‐90 mV with a pre‐pulse to 0 mV was 4.4 +/‐ 0.5 ms in control and 4.1 +/‐ 0.6 ms in ALS IgG. The relationship between the deactivation time constant and membrane potential was not significantly modified by ALS IgG. 6. ICa inactivation was not affected by ALS IgG. The potentials of half‐inactivation were ‐32.1 and ‐36.6 mV in control and ALS IgG, respectively. Similarly, the rate constants of inactivation (alpha h and beta h) remained unaltered by ALS IgG. 7. We successfully blocked ICa with 100 microM‐TMB‐8 (3,4,5‐trimethoxybenzoic acid 8‐(diethylamino)octyl ester hydrochloride), without major effects on charge movement. We adopted this procedure to study charge movement. ALS IgG reduced charge movement without significant effects on the effective valence and voltage dependence. Qon and Qoff, the charges during and after the pulse, were similarly affected by ALS IgG. 8. The actions of ALS IgG on DHP‐sensitive Ca2+ current and charge movement suggest an interaction between ALS IgG and some component of the DHP‐receptor complex.
Membrane currents in small cultured rat hippocampal neurons: a voltage‐clamp study.
Authors: Johansson S , Arhem P
Published: December 2014

1. The currents underlying the graded impulses in small cultured hippocampal neurons from rat embryos were analysed under voltage‐clamp conditions with the tight‐seal whole‐cell recording technique. 2. The leak and capacitative currents induced by a potential step were linearly related to the potential in the range studied (‐60 to ‐100 mV). 3. With steps to potentials more positive than ‐40 mV, at least two different potential‐activated currents were detected: an initial transient current and a delayed sustained one. In addition, 40% of the cells studied showed a delayed transient current. 4. The initial transient current showed sigmoid activation and roughly exponential inactivation. Its reversal potential depended on the Na+ concentration and was close to the Na+ equilibrium potential. Further, it was blocked by 3.0 microM‐tetrodotoxin, and was abolished when choline was substituted for Na+ in the extracellular solution. We concluded that this current was carried mainly by Na+ ions. 5. The delayed sustained current showed sigmoid activation and almost no inactivation within 40 ms. The reversal potential was close to the K+ equilibrium potential. We concluded that this current was carried mainly by K+ ions. 6. The delayed transient current was outward in the potential range studied (‐50 to +120 mV) and did not depend on the pipette Cl‐ concentration. It was assumed that this current was carried mainly by K+ ions. 7. A quantitative description of the initial transient and the delayed sustained currents was developed on the basis of earlier descriptions of excitable membranes.
Role of protein kinase C in constrictor responses of the rat basilar artery in vivo.
Authors: Murray M A , Faraci F M , Heistad D D
Published: December 2014

1. The goal of this study was to determine the effects of activation and inhibition of protein kinase C on the rat basilar artery in vivo. 2. The diameter of the basilar artery was measured through a craniotomy in rats anaesthetized with pentobarbitone sodium (50 mg kg‐1, I.P., supplemented with 20 mg kg‐1 h‐1). Diameters were measured under control conditions and during topical application of various agonists, both alone and in the presence of antagonists. 3. Serotonin (5‐HT) produced concentration‐related constriction of the basilar artery (baseline diameter = 234 +/‐ 9 microns, mean +/‐ S.E.M.), which was inhibited by the 5‐HT2 receptor antagonist LY53857. 4. Sphingosine (10(‐6) M), a protein kinase C inhibitor which binds to the regulatory site of protein kinase C, inhibited the response to 10(‐8) M‐serotonin (‐19 +/‐ 2% before vs. ‐3 +/‐ 2% during sphingosine, P less than 0.05). In contrast, constrictor responses to prostaglandin F2 alpha to (PGF2 alpha; 10(‐6) M) were not inhibited by sphingosine (‐16 +/‐ 2% before vs. ‐18 +/‐ 2% during sphingosine, P greater than 0.05). 5. H‐7 (10(‐9) M), another protein kinase C inhibitor, which binds to the catalytic site of protein kinase C, also inhibited constriction of the basilar artery in response to serotonin, but not prostaglandin F2 alpha. 6. Phorbol 12,13‐dibutyrate (PDBu, 10(‐8) M), which activates protein kinase C, produced slowly developing constriction of the basilar artery. PDBu‐induced vasoconstriction (‐33 +/‐ 2%) was attenuated by sphingosine (‐11 +/‐ 4% during sphingosine, P less than 0.05) and H‐7 (‐1.5 +/‐ 5% during H‐7, P less than 0.05). 7. In summary, activation of protein kinase C appears to mediate vasoconstrictor responses of the basilar artery to serotonin, but not PGF2 alpha.
Evidence that action potentials activate an internodal potassium conductance in lizard myelinated axons.
Authors: David G , Barrett J N , Barrett E F
Published: December 2014

1. We have studied action potentials and after‐potentials evoked in the internodal region of visualized lizard intramuscular nerve fibres by stimulation of the proximal nerve trunk. Voltage recordings were obtained using microelectrodes inserted into the axon (intra‐axonal) or into the layers of myelin (peri‐internodal), with the goal of studying conditions required to activate internodal K+ currents. 2. Peri‐internodal recordings made using K2SO4‐, KCl‐ or NaCl‐filled electrodes exhibited a negligible resting potential (less than 2 mV), but showed action potentials with peak amplitudes of up to 78 mV and a duration less than or equal to that of the intra‐axonally recorded action potential. 3. Following ionophoretic application of potassium from a peri‐internodal microelectrode, the peri‐internodal action potential was followed by a prolonged (hundreds of milliseconds) negative plateau. This plateau was not seen following peri‐internodal ionophoresis of sodium. The prolonged negative potential (PNP) was confined to the K(+)‐injected internode: it could be recorded by a second peri‐internodal microelectrode inserted into the same internode, but not into an adjacent internode. 4. The peri‐internodally recorded PNP was accompanied by an equally prolonged intra‐axonal depolarizing after‐potential, and by an increase in the conductance of the internodal axolemma. However, the K+ ionophoresis that produced the PNP had little or no detectable effect on the intra‐axonally or peri‐internodally recorded resting potential or action potential. These findings suggest that the PNP is generated by an inward current across the axolemma of the K(+)‐injected internode, through channels opened following the action potential. 5. Following peri‐internodal K+ ionophoresis a PNP could also be evoked by passage of depolarizing current pulses through an intra‐axonal electrode or by passage of negative current pulses through an electrode in the K(+)‐filled peri‐internodal region. The threshold for evoking a PNP was less than the threshold for evoking an action potential, and the PNP persisted in 10 microM‐tetrodotoxin. Thus the PNP is evoked by depolarization of the axolemma rather than by Na+ influx. 6. The PNP was reversibly blocked by tetraethylammonium (TEA, 2‐10 mM), but was not blocked by 100 microM‐3,4‐diaminopyridine or 5 mM‐4‐aminopyridine.(ABSTRACT TRUNCATED AT 400 WORDS)
The field adaptation of the human rod visual system.
Authors: Sharpe L T , Fach C C , Stockman A
Published: December 2014

1. Incremental thresholds were measured in a retinal region 12 deg temporal from the fovea with a target of 200 ms in duration and 6 deg in diameter superimposed on background fields of various intensities and wavelengths. Measurements were made under rod‐isolation conditions in five normal observers and in a typical, complete achromat observer who had no cone function. 2. The rise in threshold with background intensity changes with background wavelength in the normal trichromat observers. On 450, 520 and 560 nm backgrounds the average slope in logarithmic co‐ordinates (0.78 +/‐ 0.04, S.D.) is similar to that found for the achromat‐‐whose slope is independent of background wavelength (0.79 +/‐ 0.03)‐‐but on a 640 nm background it more nearly approaches Weber's law (0.91 +/‐ 0.02). This indicates that the sensitivity of the rods to an incremental target is not determined by quantal absorptions in the rods alone but by quantal absorptions in both the rods and the cones. 3. Rod incremental thresholds were also measured in various colour‐blind observers lacking one or more of the cone classes: a blue‐cone monochromat, four deuteranopes and a protanope. For the blue‐cone monochromat, like the achromat, the slope of the increment threshold curve is constant with background wavelength. For the deuteranopes and the protanope, like the normal, the slope increases with wavelength. The protanope, however, shows a smaller increase in slope, consistent with the lower sensitivity of his cones to long‐wavelength light. 4. The dependence of the field adaptation of the rods on the cones was confirmed by field‐mixture experiments, in which the incremental threshold was measured against bichromatic backgrounds, and in silent substitution experiments, in which backgrounds equated for their effects on either the cones or the rods but not both were instantaneously substituted for one another.
Imaging of intracellular calcium in rat anterior pituitary cells in response to growth hormone releasing factor.
Authors: Kato M , Hoyland J , Sikdar S K , Mason W T
Published: December 2014

1. Changes in intracellular ionized calcium [Ca2+]i induced by human growth hormone releasing factor (hGRF) were analysed by quantitative fluorescent microscopy using a dual‐wavelength, ratiometric video imaging system and low light level charge‐coupled device (CCD) camera visualizing Fura‐2 in dispersed male rat anterior pituitary cells. 2. In cells responding to hGRF, spontaneous basal oscillations in [Ca2+]i were frequently observed, and these were usually characterized by a gradient of [Ca2+]i localized in the subplasmalemmal region of the cell. 3. Of the cells which responded to hGRF, the peptide evoked a rise in [Ca2+]i, especially in the region of the subplasmalemma. Continuous application of 10 nM‐hGRF produced several different temporal patterns of the [Ca2+]i response which were not attributable to spatial response profiles. A sustained rise in [Ca2+]i was the most common type of response to hGRF (44% of the cells examined). 4. One‐third of the cells responding to 10 nM‐hGRF showed spontaneous basal [Ca2+]i oscillations ranging from 100 to 500 nM. Mean values of basal and 10 nM‐hGRF‐induced [Ca2+]i of these cells were 81 +/‐ 11 nM (mean +/‐ S.E.M., n = 27) and 560 +/‐ 47 nM (n = 27) respectively. There was no significant correlation between basal [Ca2+]i and the hGRF‐induced [Ca2+]i increase, nor was there any consistent correlation with regard to the spatial response profile. 5. Application of 2 mM‐Co2+ abolished the hGRF‐induced rise in [Ca2+]i. Quantitative analysis of this effect, performed by comparing the mean [Ca2+]i evoked during the application of hGRF with and without Co2+, respectively, also showed significant inhibition of the hGRF‐induced rise in [Ca2+]i by the application of Co2+ (P less than 0.001). 6. The hGRF‐induced rise in [Ca2+]i was completely suppressed by replacing extracellular Na+ with impermeant molecules such as mannitol. The onset and offset of suppression was as rapid as that induced by Co2+. Quantitative analysis showed significant inhibition of the hGRF‐induced rise in [Ca2+]i by Na+ replacement (P less than 0.01). 7. Tetrodotoxin, a potent blocker of voltage‐sensitive Na+ channels (5 and 20 microM), did not affect the hGRF‐induced rise in [Ca2+]i. 8. Extracellular application of the membrane permeable dibutyryl cyclic AMP (DBcAMP) to elevate intracellular levels of cyclic AMP caused a large rise in [Ca2+]i, which was dependent on extracellular Na+ and was abolished by 2 mM‐Co2+ applied in the bath.(ABSTRACT TRUNCATED AT 400 WORDS)
Tension responses to joule temperature jump in skinned rabbit muscle fibres.
Authors: Bershitsky S Y , Tsaturyan A K
Published: December 2014

1. Joule temperature jumps (T‐jumps) from 5‐9 degrees C up to 40 degrees C were used to study the cross‐bridge kinetics and thermodynamics in skinned rabbit muscle fibres. To produce a T‐jump, an alternating current pulse was passed through a fibre 5 s after removing the activating solution (pCa congruent to 4.5) from the experimental trough. The pulse frequency was congruent to 30 kHz, amplitude less than or equal to 3 kV, and duration 0.2 ms. The pulse energy liberated in the fibre was calculated using a special analog circuit and then used for estimation of the T‐jump amplitude. 2. The T‐jump induced a tri‐exponential tension transient. Phases 1 and 2 had rate constants k1 = 450‐1750 s‐1 and k2 = 60‐250 s‐1 respectively, characterizing the tension rise, whereas phase 3 had a rate constant k3 = 5‐10 s‐1 representing tension recovery due to the fibre cooling. 3. An increase from 13 to 40 degrees C for the final temperature achieved by the T‐jump led to an increase in the amplitudes of phases 1 and 2. After T‐jumps to 30‐40 degrees C during phase 1, tension increased by 50‐80%. During phase 2 an approximately 2‐fold tension increase continued. Rate constants k1 and k2 increased with temperature and temperature coefficients (Q10) were 1.6 and 1.7, respectively. 4. To study which processes in the cross‐bridges are involved in phases 1 and 2, a series of experiments were made where step length changes of ‐9 to +3 nm (hs)‐1 (nanometres per half‐sarcomere length) were applied to the fibre 4 ms before the T‐jump. 5. After the step shortening, the rate constant of phase 1 increased, whereas its amplitude decreased compared to those without a length change. This indicates that phase 1 is determined by some force‐generating process in the cross‐bridges attached to the thin filaments. This process is, most probably, the same as that producing the early tension recovery following the length change. The enthalpy change (delta H) associated with the reaction controlling this process was estimated to be positive (15‐30 kJ mol‐1). 6. Both the rate constant k2 and the maximal tension achieved at the end of phase 2 were practically independent of the preceding length changes. This means that phase 2 is accompanied by the cross‐bridge detachment and reattachment to new sites on the thin filaments.(ABSTRACT TRUNCATED AT 400 WORDS)
A novel modulatory binding site for zinc on the GABAA receptor complex in cultured rat neurones.
Authors: Smart T G
Published: December 2014

1. The properties of gamma‐aminobutyric acidA (GABAA) receptor‐ion channel complexes and the interaction with the transition metal zinc, were studied on rat sympathetic and cerebellar neurones in dissociated culture using patch clamp recording techniques. 2. The antagonism of GABA‐induced membrane currents by zinc on sympathetic neurones was subject to developmental influence. Using embryonic sympathetic neurones acutely cultured for 24‐72 h, GABA responses were more depressed by zinc when compared to responses evoked on adult neurones cultured for the same period. For neurones developing in vivo, the percentage inhibition of GABA responses produced by zinc in embryonic neurones was estimated to decline by 50% after 48.2 days following birth. 3. Embryonic sympathetic neurones maintained in culture for prolonged periods (40‐50 days in vitro, DIV) became less sensitive to zinc when compared to neurones cultured for shorter periods (10‐20 DIV). The decrease in the zinc inhibition for neurones maintained in vitro proceeded at an apparent rate of 0.55% per day. 4. Activation of the GABA receptor by muscimol (0.2‐2 microM) was also antagonized by zinc (50‐100 microM). 5. Lowering the pH of the perfusing Krebs solution did not affect the inhibition of GABA responses by zinc on sympathetic neurones. 6. Modulation of the GABAA receptor by some benzodiazepines, a barbiturate, a steroid based on pregnanolone, or antagonists bicuculline and picrotoxinin, did not interfere with the antagonism exerted by zinc on sympathetic neurones. A novel binding site for zinc on the GABAA receptor is proposed. 7. Analysis of the GABA‐activated current noise on sympathetic neurones revealed two kinetic components to the power spectra requiring a double Lorentzian fit. The time constant describing the fast component (tau 2, 2.1 ms) was unaffected by zinc, whereas the slow component time constant (tau 1, 21.7 ms) was slightly reduced to 17.1 ms. 8. The apparent single‐channel conductance for GABA‐activated ion channels was determined from the power spectra (gamma s = 22.7 pS) and also from the relationship between the mean GABA‐induced inward current and the variance of the current (gamma v = 24 pS). Zinc (25‐100 microM) did not affect the single‐channel conductance. 9. Single GABA‐activated ion channels were recorded from outside‐out patches taken from the soma of large cerebellar neurones. Single GABA channels were capable of activation to multiple current amplitudes which were assessed into the following conductance levels: 8, 18, 23, 29 and 34 pS.(ABSTRACT TRUNCATED AT 400 WORDS)
Intracellular pH regulation in isolated cochlear outer hair cells of the guinea‐pig.
Authors: Ikeda K , Saito Y , Nishiyama A , Takasaka T
Published: December 2014

1. The intracellular pH (pHi) regulation mechanisms of the outer hair cell (OHC) isolated from the guinea‐pig were studied using fluorescence ratio imaging microscopy. 2. The OHC pHi in the resting condition was 7.26 +/‐ 0.08 (mean +/‐ S.D., n = 49) when the standard solution buffered with HEPES‐Tris was superfused. 3. Exposure to 25 mM‐NH4+ in the absence of HCO3‐ caused biphasic changes in pHi; a transient increase (7.89 +/‐ 0.14, n = 22) followed by a slow decrease (7.57 +/‐ 0.12; mean +/‐ S.D.). Removal of external NH4+ by introducing the N‐methyl‐D‐glucamine (NMDG+) solution in the absence of HCO3‐ markedly acidified the pHi to 6.38 +/‐ 0.12 with little pHi recovery. Subsequent application of the standard Na+ solution restored the pHi to the initial value. The recovery was inhibited by 0.5 mM‐amiloride but not by 0.3 mM‐DIDS (4,4'‐diisothiocyanatostilbene‐2,2'‐disulphonic acid). 4. In the presence of HCO3‐, removal of both external NH4+ and Na+ promptly caused an intracellular acidification followed by a pHi recovery. The pHi recovery from an acid load was inhibited by 0.3 mM‐DIDS or 10 microM‐NPPB (5‐nitro‐2‐(3‐phenylpropyl‐amino)‐benzoate). However, the pHi in the steady state in the presence or absence of HCO3‐ was not altered by addition of 0.5 mM‐amiloride or NMDG+ solution. 5. The intracellular buffering power obtained from the NH4+ exposure and withdrawal was ‐15.1 +/‐ 8.7 mM (pH unit)‐1 (n = 6) and ‐14.3 +/‐ 5.8 mM (pH unit)‐1, respectively. 6. Replacement of external Cl‐ with gluconate in the HCO3‐ solution increased the pHi from 7.22 +/‐ 0.12 to 7.51 +/‐ 0.20 (n = 6), which was inhibited by 0.3 mM‐DIDS. Moreover, addition of DIDS to the HCO3‐ solution increased the pHi by 0.13 +/‐ 0.08 (n = 8). 7. When the external standard solution buffered with HEPES‐Tris was replaced with the HCO3‐ solution, the basal pHi (7.27 +/‐ 0.10) was promptly acidified to 6.87 +/‐ 0.10 then relaxed slowly to 7.00 +/‐ 0.15 (n = 16). 8. The pHi showed an initial alkalinization and a subsequent slow acidification after the HCO3(‐)‐free standard solution replaced the HCO3‐ solution. The slow acidification was inhibited by low external Cl‐ concentration or by addition of 0.3 mM‐DIDS.(ABSTRACT TRUNCATED AT 400 WORDS)
Adenosine promotes burst activity in guinea‐pig geniculocortical neurones through two different ionic mechanisms.
Authors: Pape H C
Published: December 2014

1. The mechanisms of action of adenosine were examined in relay neurones of the dorsal lateral geniculate nucleus (LGND) using in vitro intracellular recording techniques in guinea‐pig thalamic slices. 2. Adenosine hyperpolarized LGND relay neurones due to an increase in membrane potassium conductance. The K+ currents generated by near maximal stimulation of adenosine and GABAB receptors were non‐additive. 3. Blockage of membrane K+ conductances by barium unmasked a second response to adenosine; an outward shift of the current versus voltage relationship negative to ‐65 mV associated with an increase in membrane input resistance. The beta‐adrenoceptor agonist isoprenaline elicited an inward current in the same voltage range, which was inhibited and replaced by an outward current during activation of adenosine receptors. The effects of adenosine were due to a decrease in amplitude and rate of rise of the hyperpolarization‐activated cation current, Ih. Maximal reduction by 66% of Ih amplitude occurred near the range of half‐activation. 4. Both responses to adenosine were mimicked by the selective A1 receptor agonists N6‐cyclopentyladenosine or N6‐cyclohexyladenosine, and reversibly blocked by the selective A1 receptor antagonist 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX). 5. The decrease in Ih by adenosine may be mediated by an inhibition of adenylyl cyclase activity and hence a decrease in the intracellular level of cyclic AMP, since local application of the adenylyl cyclase inhibitor 2',3'‐dideoxyadenosine imitated the decrease in Ih. Local application of the adenylyl cyclase stimulant forskolin or 8‐bromo‐cyclic AMP resulted in an enhancement in Ih, and forskolin inhibited the action on Ih evoked by N6‐cyclopentyladenosine. 6. The adenosine‐induced effects interacted with the intrinsic electrophysiological properties of LGND neurones in that (i) the hyperpolarization due to an increase in K+ conductance inhibited single spike firing and promoted calcium‐mediated burst discharges, and (ii) the decrease in Ih inhibited the dampening effect on Ca(2+)‐mediated rebound activity of beta‐adrenergic receptor stimulation. 7. It is suggested that during increased levels of extracellular adenosine the response of LGND relay neurones to activating brainstem influences will be depressed, and a pattern of Ca(2+)‐mediated burst firing will be favoured.
Cyclic GMP‐activated channels of salamander retinal rods: spatial distribution and variation of responsiveness.
Authors: Karpen J W , Loney D A , Baylor D A
Published: December 2014

1. Patch‐clamp methods were used to investigate the areal density and spatial location of cyclic GMP‐activated channels in the surface membrane of salamander rod outer segments. 2. The density of active channels (i.e. channels able to respond to cyclic GMP) in patches excised from outer segments was determined from the number of active channels, N, and the membrane area, A. N was estimated from the current induced by a saturating concentration of cyclic GMP, while A was estimated from the electrical capacitance of the patch. 3. In patches excised from forty‐one isolated outer segments prepared in the light the active channel density varied over a remarkable range: 0.34‐629 microns‐2, with a mean of 166 microns‐2. Density was not correlated with patch area in this or any of the conditions studied. 4. The spatial distribution of open channels on the outer segment of a transducing rod was measured by recording the local dark current at various positions with a loose‐patch electrode. The apparent density of open channels varied by only about +/‐ 50% around the circumference of the outer segment and up and down its length. This indicates that the wide range of densities in excised patches did not result from sampling a non‐uniform spatial distribution of channels. 5. Patches excised from sixteen dark‐adapted whole cells with healthy appearances and saturating light responses of normal size had active channel densities of 1.1‐200 microns‐2, with a mean of 60 microns‐2. Patches from twenty light‐adapted whole cells had similar densities. Many densities from the whole cells were much lower than expected. This, and the wide variation in densities, suggests that obtaining a patch often lowered the density of active channels. The number of channels in a patch was quite stable from 1 s to 30 min after excision, ruling out progressive denaturation or adsorption of channels to the glass as a cause for this effect. 6. The mean active channel density in patches excised from whole cells was lower with calcium present in the external solution than with calcium absent (80 vs. 152 microns‐2, n = 36 and 30 respectively). 7. We conclude that copies of the channel protein were present at a density of at least 650 microns‐2 in the surface membrane of the outer segment and that the distribution of channels was fairly uniform on a 1 micron scale.(ABSTRACT TRUNCATED AT 400 WORDS)
The pH dependence of chloride net flux in skeletal muscle fibres of Rana temporaria.
Authors: Hansen M , Skydsgaard J M
Published: December 2014

1. The pH‐ and voltage‐dependent Cl‐ conductance in frog muscle may be related to the chloride equilibrium potential, VCl,eq, rather than to the absolute membrane potential, Vm. This hypothesis was tested in thin depolarized frog muscle fibre bundles by investigating the influence of pH on 36Cl‐ efflux during Cl‐ net efflux upon removal of external Cl‐, which offers a state far from Cl‐ equilibrium by changing VCl,eq instead of Vm. 2. Upon the change from Cl‐ equilibrium at 20 mM‐Clo‐ to Cl‐ net efflux at zero [Cl‐]o, Vm changed only approximately 10 mV, but the 36Cl‐ efflux rate increased about three times at pH 5.5 and decreased to less than one tenth of the equilibrium efflux rate at pH 9.8. The switch between ‘acid’ and ‘alkaline’ responses occurs at a pH between 6.4 (‘acid response’) and 7.2 (‘alkaline response’). 3. Changing pH between 5.5 and 9.8 during Cl‐ net efflux showed an increase of 36Cl‐ net flux rate upon acidification and a decrease upon alkalinization. The reactions are opposite to those seen by pH shifts at chloride equilibrium. 4. The changes of net flux rate coefficients upon changes of pH were transient, especially the activation at low pH that relaxed significantly during about 10 min. 5. The results are consistent with the notion that the state of the gCl mechanism in frog muscle is related to (Vm‐VCl,eq) rather than to the absolute potential alone.
Response characteristics of the cells of cortical area 21a of the cat with special reference to orientation specificity.
Authors: Wimborne B M , Henry G H
Published: December 2014

1. Extracellular recording using tungsten‐in‐glass microelectrodes was conducted on 115 neurons in area 21a of fifteen anaesthetized cats. Quantitative analysis using computer‐controlled display and collecting routines were used to investigate the excitatory and inhibitory regions of the receptive field and to see if interaction, within and between these regions, contributed to the response properties of the cells. 2. The responses of the cells in the sample appeared to arise from a single, homogeneous class. All cells had single discharge regions which responded with composite ON/OFF firing to a stationary flashing bar. The same region also responded to moving light and dark bars and edges. There was little evidence of inhibition as measured by the suppression of spontaneous or induced firing. Most cells had relatively small receptive fields (primary width: mean = 2.1 +/‐ 0.9 deg (S.D.); n = 108), all were binocular and were located within 15.0 deg of the visual axes. 3. All cells responded well to slowly moving stimuli but generally failed to respond to stimuli moving faster than 10.0 deg s‐1. All responses were bi‐directional and, although many showed evidence of length summation, there was no sign of linear summation. 4. Despite the absence of significant sideband inhibition many cells were acutely tuned for orientation (half‐width at half‐height: mean = 15.6 +/‐ 5.3 deg; n = 48). To investigate this property further, cells were analysed to assess the effect of changing the length of a moving bar stimulus on the acuteness of the orientation tuning curve. Short bars, of similar length to the width of the receptive field, had orientation tuning curves of equivalent sharpness to those obtained with longer bars. 5. The equivalence of orientation tuning for long and short bars stands in contrast to the results obtained for both simple (S) and complex (C) cells of the striate cortex where tuning for the longer bar is sharper than that for the shorter. The result from area 21a cells is consistent with the absence of sideband inhibition and can be related to an input from the striate cortex that passes through a threshold barrier. 6. The orientation tuning of cells of area 21a can be explained if it is assumed that they receive their major input from C or complex cells of the striate cortex in which firing must reach a threshold frequency to activate the recipient cell.
Direct modulation of GABAA receptor by intracellular ATP in dissociated nucleus tractus solitarii neurones of rat.
Authors: Shirasaki T , Aibara K , Akaike N
Published: December 2014

1. Effect of intracellular ATP on Cl‐ current (ICl) mediated by the GABA (gamma‐aminobutyric acid) receptor subtype, GABAA, was studied in dissociated nucleus tractus solitarii (NTS) neurones using the whole‐cell mode of patch clamp. A concentration‐jump technique termed ‘Y tube’ was used to rapidly apply agents externally. Dissociated neurones were obtained from 1‐ to 3‐week‐old rats. 2. When the patch‐pipette solution contained 2 mM‐ATP, the amplitude of ICl elicited by 10(‐5) M‐GABA did not show any time‐dependent decrease (apparent run‐down), for more than 60 min after the initial recording. In the presence of ATP, the half‐maximum concentration (KD) and Hill coefficient calculated from the GABA concentration‐response curve were 9.12 microM and 1.47, respectively. 3. In the absence of intracellular ATP, the amplitude of GABA‐induced ICl decreased with time. The relative peak amplitudes after 20 and 60 min from the initial recording were 0.40 +/‐ 0.09 (n = 11) and 0.16 +/‐ 0.05 (n = 8) with respect to the initial response. 4. Removal of Mg2+ from the internal solution induced run‐down of the GABA response even in the presence of 2 mM‐intracellular ATP, suggesting that both intracellular ATP (2 mM or more) and Mg2+ are necessary to prevent run‐down of the GABA response. 5. Activation of dephosphorylation processes by alkaline phosphatase (100‐200 microM) did not affect the GABA response in neurones perfused with internal solution containing 2 mM‐ATP and 3 mM‐Mg2+. Blocking the dephosphorylation process by okadaic acid, a phosphatase inhibitor, did not prevent the run‐down of the GABA response. 6. Calcium influxes passing through both the voltage‐dependent L‐type Ca2+ channel and the glutamate receptor‐operated cation channel did not affect ICl induced by GABA. 7. GABA‐induced ICl was also maintained by adding 2 mM‐ADP or ATP gamma S (adenosine‐5'‐O‐3‐thiotriphosphate) to the internal solution containing Mg2+. Addition of 2 mM‐adenosine, AMP, cyclic AMP, AMP‐PNP (adenylimido‐diphosphate) or ADP beta S (adenosine‐5'‐O‐2‐thiodiphosphate) to the internal solution did not prevent the run‐down of the GABA response even in the presence of 3 mM‐intracellular Mg2+. Based on the chemical specificity of these ATP analogues, it is suggested that there is an ATP‐sensitive binding site (ATP receptor) in the cytoplasmic side of the cell membrane.(ABSTRACT TRUNCATED AT 400 WORDS)
Alveolar epithelial permeability in baboons: histamine and capsaicin.
Authors: Yeates D B , Hameister W M
Published: December 2014

1. We investigated if changes in alveolar epithelial permeability could be initiated by various doses of the irritant capsaicin through stimulation of pulmonary afferent nerves either directly, or indirectly through the release of an intermediate cell mediator, and whether mediation of permeability involved histamine H1 and/or H2 receptors. 2. Alveolar epithelial permeability was indicated by the clearance of peripherally deposited 99mTc‐DTPA (technetium‐99m‐diethylenetriamine pentaacetate) aerosol in baboons. Aerosol challenge experiments were performed twice with histamine (32 mg/ml), once each with capsaicin (10(‐6), 10(‐5), 10(‐4) and 10(‐3) M), and once each with histamine (32 mg/ml) preceded by H1 (terfenadine) and H2 (ranitidine) antagonists alone and combined. 3. Mean half‐time for 99mTc‐DTPA clearance was 66 +/‐ 4 min. After histamine, it decreased to 41 +/‐ 5 min (P < 0.05), and after capsaicin it was 70 +/‐ 5 min (combined doses). Pretreatment with the H1 and H2 antagonists, either separately or in combination, did not consistently inhibit increases in 99mTc‐DTPA permeability. 4. Capsaicin caused tachypnoea only at 10(‐3) M (P < 0.05). Inhibition of histamine‐induced tachypnoea required both antagonists (P < 0.05) suggesting the existence of a population of peripheral sensory neurons possessing H1 and H2 receptors. Changes in permeability were unrelated to the changes in respiratory frequency. Thus, neither the mechanisms that stimulate respiratory frequency, nor the respiratory frequency per se, were responsible for increased alveolar epithelial permeability. 5. Capsaicin did not change epithelial permeability at doses that cause capsaicin‐sensitive C fibre stimulation as delineated by increases in respiratory frequency. Nor does C fibre stimulation, induced by mediators released from pulmonary cells activated by low doses of capsaicin, increase epithelial permeability. These data suggest that in the primate, histamine increases epithelial permeability to small solutes by a mechanism independent of the activation of capsaicin‐sensitive C fibres and capsaicin‐initiated release of cell mediators. 6. These findings do not support a role for C fibre activation in regulating epithelial permeability of small solutes.
Serotonin regulates the phase of the rat suprachiasmatic circadian pacemaker in vitro only during the subjective day.
Authors: Medanic M , Gillette M U
Published: December 2014

1. The suprachiasmatic nucleus (SCN) of the hypothalamus is the primary pacemaker for circadian rhythms in mammals. The 24 h pacemaker is endogenous to the SCN and persists for multiple cycles in the suprachiasmatic brain slice. 2. While serotonin is not endogenous to the SCN, a major midbrain hypothalamic afferent pathway is serotonergic. Within this tract the dorsal raphe nucleus sends direct projections to the ventrolateral portions of the SCN. We investigated a possible regulatory role for serotonin in the mammalian circadian system by examining its effect, when applied at projection sites, on the circadian rhythm of neuronal activity in rat SCN in vitro. 3. Eight‐week‐old male rats from our inbred colony, housed on a 12 h light: 12 h dark schedule, were used. Hypothalamic brain slices containing the paired SCN were prepared in the day and maintained in glucose and bicarbonate‐supplemented balanced salt solution for up to 53 h. 4. A 10(‐11) ml drop of 10(‐6) M‐serotonin (5‐hydroxytryptamine (5‐HT) creatinine sulphate complex) in medium was applied to the ventrolateral portion of one of the SCN for 5 min on the first day in vitro. The effect of the treatment at each of seven time points across the circadian cycle was examined. The rhythm of spontaneous neuronal activity was recorded extracellularly on the second and third days in vitro. Phase shifts were determined by comparing the time‐of‐peak of neuronal activity in serotonin‐ vs. media‐treated slices. 5. Application of serotonin during the subjective day induced significant advances in the phase of the electrical activity rhythm (n = 11). The most sensitive time of treatment was CT 7 (circadian time 7 is 7 h after ‘lights on’ in the animal colony), when a 7.0 +/‐ 0.1 h phase advance was observed (n = 3). This phase advance was perpetuated on day 3 in vitro without decrement. Serotonin treatment during the subjective night had no effect on the timing of the electrical activity rhythm (n = 9). 6. The specificity of the serotonin‐induced phase change was assessed by treating slices in the same manner with a microdrop of serotonergic agonists, 5‐carboxamidotryptamine, that targets the 5‐HT1 class of receptors, or 8‐hydroxy‐dipropylaminotetralin (8‐OH DPAT), that acts on the 5‐HT1A receptor subtype.(ABSTRACT TRUNCATED AT 400 WORDS)