bluuebunny/crossref_metadata_2025 huggingface dataset:
[Ca2+]i transients and [Ca2+]i‐dependent chloride current in single Purkinje cells from rabbit heart.
Authors: Sipido K R , Callewaert G , Carmeliet E
Published: December 2014

1. Single Purkinje cells, enzymatically isolated from rabbit ventricle, were studied under whole‐cell voltage clamp and internally perfused with the fluorescent Ca2+ indicator, indo‐1 (100 microM). 2. Fast [Ca2+]i transients were elicited by brief depolarizations from a holding voltage of ‐45 mV and by repolarization from very positive potentials. The peak [Ca2+]i‐voltage relation was bell‐shaped with a peak around +10 mV. 3. [Ca2+]i transients were completely blocked by the Ca2+ channel antagonist, nisoldipine (10 microM) and were very small when Ca2+ release from the sarcoplasmic reticulum (SR) was prevented by superfusion of cells by caffeine (1 mM) or ryanodine (10 microM). A fast application of caffeine induced a transient increase in [Ca2+]i. These results suggest [Ca2+]i transients are due to Ca(2+)‐induced Ca2+ release from the SR. 4. Rate of decline of the [Ca2+]i transient was voltage dependent, suggesting contribution of the Na(+)‐Ca2+ exchanger to Ca2+ efflux. At very positive potentials (> +60 mV), Ca2+ influx through the Na(+)‐Ca2+ exchanger could be observed. 5. A transient outward current was observed at potentials positive to +10 mV, but only if depolarizing pulses were accompanied by a [Ca2+]i transient. 6. When the amplitude of the [Ca2+]i transient was changed by (1) changes in [Ca2+]o, (2) changes in frequency of depolarization or (3) conditioning prepulses, the amplitude of the outward current changed in the same direction. This suggests activation of the current is dependent on and graded by [Ca2+]i. 7. The outward current was observed in K(+)‐free solutions, in the presence of Cs+ and TEA+, and was not blocked by 4‐aminopyridine (10 mM). In contrast, DIDS (100 microM) decreased the outward current by 70 +/‐ 20% (mean +/‐ S.D., n = 9), without affecting [Ca2+]i. 8. When external Cl‐ was lowered, the amplitude of the outward current decreased; when internal Cl‐ was replaced by aspartate, it became apparent at more negative potentials. These interventions strongly suggest the current was carried by Cl‐; it can therefore be referred to as a [Ca2+]i‐activated Cl‐ current or ICl(Ca). 9. When ICl(Ca) was maximally activated during a conditioning step, steps to negative potentials revealed inward currents through ICl(Ca) (in symmetrical Cl‐ solutions). The fully activated I‐V relation was linear. 10. ICl(Ca) could be activated at membrane potentials between ‐80 and +80 mV by a fast application of caffeine (10 mM), inducing Ca2+ release from the SR, demonstrating that ICl(Ca) does not require membrane depolarization or Ca2+ influx through the Ca2+ channel for its activation.(ABSTRACT TRUNCATED AT 400 WORDS)
The contribution of [Ca2+]i to the slowing of relaxation in fatigued single fibres from mouse skeletal muscle.
Authors: Westerblad H , Allen D G
Published: December 2014

1. The contribution of Ca2+ vs. cross‐bridges to the slowing of relaxation in fatigue was studied in intact, single fibres dissected from a mouse foot muscle. Fatigue was produced by repeated 350 ms tetani. The free myoplasmic Ca2+ concentration ([Ca2+]i) was measured with indo‐1. 2. The rate of [Ca2+]i decline after a tetanus was reduced in fatigue but this reduction appeared smaller than the decrease in relaxation speed. 3. Under control conditions, steady‐state [Ca2+]i‐tension curves were constructed from measurements of tetani at various stimulus frequencies. Measurements from the final part of fatigue runs, where both tetanic [Ca2+]i and tension fell relatively fast, were used to construct similar curves in fatigue. A comparison of these curves revealed a reduction of both the maximum Ca(2+)‐activated tension and the myofibrillar Ca2+ sensitivity in fatigue. 4. Calcium‐derived tension records were generated by converting [Ca2+]i signals during tetani into tension by means of the steady‐state [Ca2+]i‐tension relation. These records would represent a situation where tension responds to changes of [Ca2+]i without delays due to, for instance, cross‐bridge attachment or detachment. The relaxation speed of the calcium‐derived tension was similar in control and fatigue; the lag between the calcium‐derived and the real tension was about twice as long in fatigue. 5. Both the decline of [Ca2+]i and tension were markedly faster after a 10 s pause elicited during fatigue runs at the time of maximum slowing of relaxation. The relaxation of the calcium‐derived tension was also faster after the pause. 6. The function of the sarcoplasmic reticulum (SR) Ca2+ pumps was analysed by plotting the rate of [Ca2+]i decline vs. [Ca2+]i during the tail of elevated [Ca2+]i after tetani. This analysis showed approximately a sevenfold reduction of the pump rate in fatigue. 7. In conclusion, the rate of [Ca2+]i decline after a tetanus is reduced in fatigue probably due to impaired SR Ca2+ pumping. This is not the cause of the slowed relaxation because it is counteracted by a reduced myofibrillar Ca2+ sensitivity. Thus, the slowing of relaxation in fatigued mouse muscle fibres would reflect slowed cross‐bridge kinetics.
An ionic current model for neurons in the rat medial nucleus tractus solitarii receiving sensory afferent input.
Authors: Schild J H , Khushalani S , Clark J W , Andresen M C , Kunze D L , Yang M
Published: December 2014

1. Neurons from a horizontal slice of adult rat brainstem were examined using intracellular recording techniques. Investigations were restricted to a region within the nucleus tractus solitarii, medial to the solitary tract and centred on the obex (mNTS). Previous work has shown this restricted area of the NTS to contain the greatest concentration of aortic afferent baroreceptor terminal fields. Electrical stimulation of the tract elicited short‐latency excitatory postsynaptic potentials in all neurons. 2. mNTS neurons were spontaneously active with firing frequencies ranging between 1 and 10 Hz, at resting potentials of ‐65 to ‐45 mV. These neurons did not exhibit spontaneous bursting activity. 3. Depolarizing current injection immediately evoked a finite, high‐frequency spike discharge which rapidly declined to a lower steady‐state level (i.e. spike frequency adaptation, SFA). Increasing depolarizations produced a marked increase in the peak instantaneous frequency but a much smaller increase in the steady‐state firing level. 4. Conditioning with a hyperpolarizing prepulse resulted in a prolonged delay of up to 600 ms before the first action potential (i.e. delayed excitation, DE) with an attendant decrease in peak discharge rates. DE was modulated by both the magnitude and duration of the prestimulus hyperpolarization, as well as the magnitude of the depolarizing stimulus. Tetrodotoxin (TTX) eliminated spike discharge but had little effect on the ramp‐like membrane depolarization characteristic of DE. 5. We have developed a mathematical model for mNTS neurons to facilitate our understanding of the interplay between the underlying ionic currents. It consists of a comprehensive membrane model of the Hodgkin‐Huxley type coupled with a fluid compartment model describing cytoplasmic [Ca2+]i homeostasis. 6. The model suggests that (a) SFA is caused by an increase in [Ca2+]i which activates the outward K+ current, IK,Ca, and (b) DE results from the competitive interaction between the injected depolarizing current and the hyperpolarization‐activated transient outward K+ currents, IA and ID. 7. We conclude that our ionic current model is capable of providing biophysical explanations for a number of phenomena associated with brainstem neurons, either during spontaneous activity or in response to patterned injections of current. This model is a potentially useful adjunct for on‐going research into the central mechanisms involved in the regulation of both blood pressure and ventilation.
GLUT 4 and insulin receptor binding and kinase activity in trained human muscle.
Authors: Dela F , Handberg A , Mikines K J , Vinten J , Galbo H
Published: December 2014

1. Physical training enhances sensitivity and responsiveness of insulin‐mediated glucose uptake in human muscle. This study examines if this effect of physical training is due to increased insulin receptor function or increased total concentration of insulin‐recruitable glucose transporter protein (GLUT 4). 2. Seven healthy young subjects carried out single leg bicycle training for 10 weeks at 70% of one leg maximal oxygen uptake (VO2,max). Subsequently biopsies were taken from the vastus lateralis muscle of both legs. 3. Single leg VO2,max increased for the trained leg (46 +/‐ 3 to 52 +/‐ 2 ml min‐1 kg‐1 (means +/‐ S.E.M., P < 0.05), and cytochrome c oxidase activity was higher in this compared to the untrained leg (2.0 +/‐ 0.1 vs. 1.4 +/‐ 0.1 nmol s‐1 (mg muscle)‐1, P < 0.05). Insulin binding as well as basal‐ and insulin‐stimulated receptor kinase activity did not differ between trained and untrained muscle. The concentration of GLUT 4 protein was higher in the former (14.9 +/‐ 1.9 vs. 11.6 +/‐ 1.0 arbitrary units (micrograms protein)‐1 in crude membranes, P < 0.05). The training‐induced increase in GLUT 4 (26 +/‐ 11%) matched a previously reported increase in maximum insulin‐stimulated leg glucose uptake (25 +/‐ 7%) in the same subjects, and individual values of the two variables correlated (correlation coefficient (r) = 0.84, P < 0.05). 4. In conclusion, in human muscle training induces a local contraction‐dependent increase in GLUT 4 protein, which enhances the effect of insulin on glucose uptake. On the other hand, insulin receptor function in muscle is unlikely to be affected by training.
GABAB receptor modulation of Ca2+ currents in rat sensory neurones by the G protein G(0): antisense oligonucleotide studies.
Authors: Campbell V , Berrow N , Dolphin A C
Published: December 2014

1. Calcium channel currents (IBa) were recorded in cultured dorsal root ganglion neurones (DRGs), 24‐32 h after microinjection with 20‐mer phosphorothioate antisense oligonucleotides complementary either to a G alpha o or a G alpha i unique sequence, or with a nonsense sequence. 2. The ability of the GABAB agonist (‐)‐baclofen (50 microM) to inhibit IBa was examined. The maximum peak current was inhibited by 35.3 +/‐ 4.0% (n = 11) in control non‐injected cells, and by 38.1 +/‐ 2.6% (n = 11) and 34.8 +/‐ 4.2% (n = 5) in nonsense‐ and G alpha i oligonucleotide‐injected cells. Following G alpha o oligonucleotide injection, (‐)‐baclofen inhibited IBa by 21.0 +/‐ 3.2% (n = 19). 3. Confocal immunocytochemical localization of G alpha o showed prominent staining at the plasma membrane in control DRGs, and this was also present in G alpha i and nonsense oligonucleotide‐injected cells. The G alpha o staining at the plasma membrane was reduced by 76% in G alpha o oligonucleotide‐injected cells. In contrast, confocal immunocytochemical localization of G alpha i showed immunostaining in the membrane and cytoplasm of control, G alpha o‐ and nonsense‐injected DRGs, whereas this was depleted by 68% in G alpha i oligonucleotide‐injected cells. 4. These results indicate that the GABAB receptor couples to voltage‐sensitive calcium channels via the G protein G(o) and not Gi, and that antisense oligonucleotides can be used to deplete G proteins in DRGs.
A voltage‐dependent proton current in cultured human skeletal muscle myotubes.
Authors: Bernheim L , Krause R M , Baroffio A , Hamann M , Kaelin A , Bader C R
Published: December 2014

1. A voltage‐dependent proton current, IH, was studied in cultured myotubes obtained from biopsies of human muscle, using whole‐cell recording with the patch‐clamp technique. 2. With a pHo of 8.0 and a calculated pHi of 6.3, IH was activated at voltages more depolarized than ‐50 mV and its conductance reached its maximum value at voltages more depolarized than +10 mV. 3. Studies of the reversal potential of IH during substitution of K+, Na+, Ca2+, Cl‐, Cs+ and H+ in the extracellular solution indicated that protons were the major charge carriers of IH. 4. IH was also activated during a voltage step to +22 mV with a pHo of 7.3 and a calculated pHi of 7.3. 5. Acidification of the extracellular solution led to a shift towards depolarized voltages of the conductance‐voltage relationship. 6. Stationary noise analysis of IH suggested that the elementary event underlying IH was very small with a conductance of less than 0.09 pS. 7. Extracellular application of various divalent cations blocked IH. The block by divalent cations was voltage dependent, being more efficient at hyperpolarized than at depolarized voltages. For Cd2+, the Michaelis‐Menten constant (Km) for the block was 0.6 microM at ‐28 mV and 10.4 microM at +12 mV. 8. Ca2+ was a less efficient blocker than Cd2+ but could block IH at physiological concentrations (the Km values for the block were 0.9 mM at ‐38 mV and 7.3 mM at ‐8 mV). 9. The voltage‐dependent properties of IH and its ability to be affected by pH and Ca2+ suggest that IH might be used by skeletal muscle cells to extrude protons during action potentials. 10. A model of IH activation suggests that under extreme conditions, the conductance of IH can reach 40% of its maximum value after less than ten action potentials.
Inward current activated by carbachol in rat intestinal smooth muscle cells.
Authors: Ito S , Ohta T , Nakazato Y
Published: December 2014

1. Carbachol (0.1 mM or 10 microM)‐evoked inward currents were studied with standard and perforated whole‐cell patch clamp techniques in smooth muscle cells isolated from rat small intestine. The intracellular free Ca2+ concentration was monitored simultaneously with the fura‐2 method. 2. With a K(+)‐containing pipette solution, carbachol produced an inward current at ‐60 mV and a large outward current at ‐20 mV. 3. When NaCl was substituted for KCl in the external and pipette solutions, carbachol elicited inward currents at holding potentials more inside‐negative than 0 mV. The reversal potential of the carbachol‐induced current altered when external chloride (‐0.9 mV) was replaced by iodide (‐21.2 mV), thiocyanate (‐27.0 mV) and glutamate (18.2 mV). The carbachol‐induced current at ‐60 mV was slightly decreased by the replacement of external NaCl with Tris‐Cl. 4. The carbachol‐induced inward current at ‐60 mV was accompanied by an increase in the intracellular concentration of free Ca2+. Both responses to carbachol were observed 2 min after exposure of the cells to a Ca(2+)‐free solution containing 2 mM EGTA. 5. Intracellular application of heparin inhibited the inward current and Ca2+ transient responses to carbachol but not those to caffeine (10 mM). An inward current and Ca2+ transient were elicited after the patch membrane was ruptured at ‐60 mV, using a patch pipette containing inositol 1,4,5‐trisphosphate (InsP3). 6. It is concluded that the carbachol‐induced inward current is due to increases in membrane Cl‐ and Na+ conductances. Ca2+ released from InsP3‐sensitive stores may play a role in increasing both conductances.
The role of vasopressin in the regional vascular responses evoked in the spontaneously breathing rat by systemic hypoxia.
Authors: Louwerse A M , Marshall J M
Published: December 2014

1. In spontaneously breathing rats anaesthetized with Saffan, we have investigated the role of vasopressin in the cardiovascular responses evoked by systemic hypoxia (breathing 8 or 6% O2 for 5 min). 2. Breathing 8% O2 evoked an increase in respiratory frequency and tidal volume; arterial O2 pressure (Pa,O2) fell to 37 mmHg and arterial CO2 pressure (Pa,CO2) fell to 30 mmHg. Concomitantly, there was a fall in arterial pressure, tachycardia and increases in femoral and renal vascular conductances indicating net vasodilatation in skeletal muscle and kidney. The vasopressin V1‐receptor antagonist, d(CH2)5Tyr(Me)‐arginine vasopressin (20 micrograms kg‐1 i.v.), had no significant effect on the baseline values of any recorded variables, nor on the respiratory or blood gas changes evoked by 8% O2. However, it accentuated the fall in arterial pressure and the increase in femoral vascular conductance (+22 vs. +77% at the 5th minute) produced by 8% O2, but had no significant effect on the increase in renal vascular conductance. 3. Breathing 6% O2 evoked qualitatively similar responses as 8% O2 but Pa,O2 fell to 33 mmHg and Pa,CO2 fell to 28 mmHg and the respiratory and cardiovascular changes tended to be larger than those evoked by 8% O2. Again the V1‐receptor antagonist accentuated the hypoxia‐induced fall in arterial pressure and increase in femoral vascular conductance (+5 vs. +76% at the 5th minute). 4. Infusion of vasopressin (1.5 ng min‐1 kg‐1 i.v.) for 5 min with the aim of producing a plasma concentration comparable to that reached during 8% O2, induced a rise in arterial pressure (9%), bradycardia (‐5%) and a decrease in femoral (‐11%) and renal vascular conductance (‐4%). 5. These results suggest that vasopressin released during hypocapnic hypoxia helps to limit the evoked fall in arterial pressure by exerting a vasoconstrictor influence on skeletal muscle.
Role of GTP‐protein and endothelium in contraction induced by ethanol in pig coronary artery.
Authors: Kuroiwa M , Aoki H , Kobayashi S , Nishimura J , Kanaide H
Published: December 2014

1. We examined the effects of ethanol on the contractility of strips of porcine coronary artery, with and without endothelium, and following permeabilization with alpha‐toxin, and of aortic valvular endothelial cells, in situ. Changes in cytosolic Ca2+ concentration ([Ca2+]i) of the coronary artery smooth muscle cells and of the valvular endothelial cells were monitored using front‐surface fluorometry of the calcium indicator dye, fura‐2. In permeabilized preparations, [Ca2+]i was clamped using 10 mM ethyleneglycol‐bis‐(beta‐aminoethylether)‐N,N,N',N'‐tetra ace tic acid (EGTA) and 10 microM A23187 (a calcium ionophore). 2. The strips without endothelium were placed in normal physiological salt solution (normal PSS) in the presence of ethanol (100‐1000 mM). There were dose‐dependent increases in [Ca2+]i and a rapid sustained rise in tension. In Ca(2+)‐free PSS, ethanol increased [Ca2+]i and tension, similar to, but much smaller than, findings with normal PSS. 3. For a given change in [Ca2+]i induced by ethanol, the developed tension was greater than that observed during contractions induced by high [K+]o. Thus, the [Ca2+]‐tension curve for ethanol was shifted to the left of that for high [K+]o. The [Ca2+]‐tension curve for the contraction induced by ethanol in the absence of extracellular Ca2+ was shifted further to the left from that obtained in the presence of [Ca2+]o. 4. The mechanisms involved in this Ca(2+)‐sensitizing effect of ethanol were investigated using alpha‐toxin‐permeabilized coronary medial strips. Ethanol increased the tension development, in a concentration‐dependent manner, at a fixed concentration of Ca2+ (pCa = 6.3) in the presence of guanosine‐5'‐triphosphate (GTP), an effect antagonized by guanosine‐5'‐O‐(beta‐thiodiphosphate) (GDP beta S), a non‐hydrolysable GDP analogue. 5. With intact endothelium, the ethanol‐induced tension development was markedly reduced, although inhibition in the increase in [Ca2+]i was slight. The [Ca2+]‐tension relationship of this contraction overlapped with that obtained with high [K+]o‐induced contraction and was shifted to the right from that obtained in the absence of the endothelium. This endothelium‐dependent reduction of [Ca2+]i and tension induced by ethanol was inhibited when the strips were exposed to NG‐monomethyl‐L‐arginine (L‐NMMA). 6. Ethanol induced a gradual and sustained increase in [Ca2+]i in normal PSS, and a transient, concentration‐dependent increase in [Ca2+]i in Ca(2+)‐free PSS in porcine aortic valvular endothelial cells in situ.(ABSTRACT TRUNCATED AT 400 WORDS)
Somatostatin‐mediated inhibitory postsynaptic potential in sympathetically denervated guinea‐pig submucosal neurones.
Authors: Shen K Z , Surprenant A
Published: December 2014

1. Intracellular recordings were made from submucosal neurones in guinea‐pig ileum. In some animals, the extrinsic (sympathetic) nerves to the submucosal plexus were severed 5‐7 days previously. The actions of somatostatin and somatostatin analogues on membrane potential, membrane current and inhibitory postsynaptic potentials (IPSPs) were examined. 2. Somatostatin, somatostatin(1‐28), [D‐Trp8]somatostatin and the somatostatin analogue CGP 23996 all produced equivalent maximum hyperpolarizations or outward currents; half‐maximal concentrations (EC50 values) were 9‐11 nM. The somatostatin analogue MK 678 had an EC50 of 0.9 nM. Extrinsic sympathectomy did not alter concentration‐response relations for somatostatin or its analogues. 3. Somatostatin (> 100 nM) produced hyperpolarization or outward current that declined almost completely during superfusion for 2‐4 min; decline of the somatostatin current was exponential with a time constant of 30 s in the presence of 2 microM somatostatin. Desensitization was not altered by extrinsic denervation. 4. Recovery from desensitization was rapid and followed the time course of agonist wash‐out. Forskolin, phorbol esters, dithiothreitol, hydrogen peroxide, concanavalin A, or reducing temperature from 35 to 29 degrees C did not alter the time course, degree of, or recovery from desensitization. 5. The somatostatin‐induced desensitization was of the homologous type; no cross‐desensitization to opiate or alpha 2‐adrenoceptor agonists (which activate the same potassium conductance) occurred. 6. Somatostatin desensitization did not alter the adrenergic IPSP seen in sympathetically innervated preparations but abolished the non‐adrenergic IPSP recorded from normal preparations and from preparations in which the extrinsic sympathetic nerve supply had been surgically removed. 7. The selective blockade of the non‐adrenergic IPSP by the homologous‐type somatostatin desensitization characterized in the present study provides strong support for the hypothesis that somatostatin is the neurotransmitter underlying the non‐adrenergic IPSP in both normal and extrinsically denervated submucosal neurones.
Evidence for multiple open states of the Ca2+ channels in smooth muscle cells isolated from the guinea‐pig detrusor.
Authors: Nakayama S , Brading A F
Published: December 2014

1. Whole‐cell voltage clamp techniques were used to examine the properties of voltage‐dependent Ca2+ channel currents in single smooth muscle cells enzymatically dissociated from guinea‐pig urinary bladder. Potassium currents were blocked with intracellular Cs+. A holding potential of ‐60 mV was normally applied. 2. When the membrane potential was returned to the holding potential after a depolarizing step, tail currents were seen after depolarizations to positive potentials, and the size of the tail current increased with increasing positivity of the preceding depolarization. 3. After depolarization to +80 mV (a potential at which little inward current flowed through the Ca2+ channels) tail currents on returning to the holding potential increased in size as the duration of the depolarization was increased. 4. Investigation of the mechanism mediating the tail currents showed that they were not flowing through non‐selective cation channels, and had no contribution from Ca(2+)‐activated Cl‐ channels or Na(+)‐Ca2+ exchange. 5. The tail currents and the inward currents evoked by a simple depolarizing test potential were equally decreased by nifedipine in a dose‐dependent manner. This suggests that L‐type Ca2+ channels are responsible for both of the two types of inward currents. The inward currents were also inhibited in a similar manner when caffeine was applied. 6. Although the tail currents evoked on stepping from +80 mV to a holding potential of ‐60 mV increased in size with the duration of the conditioning potential, the total membrane Ca2+ conductance did not increase, since the inward currents evoked on stepping to +20 mV (a potential at which the Ca2+ channels are still fully activated) did not change with time. 7. The amplitude of the inward current evoked by a simple depolarizing test potential was similar to that evoked on stepping to the same test potential after preconditioning at +80 mV, if the test potential was higher than +20 mV. However, following repolarization to the holding potential, the amplitude of the subsequent tail current was larger and the deactivation time constant longer, after the conditioning depolarization. These results suggest that the voltage‐dependent Ca2+ channels have at least two open states with different time constants, the tail current being the result of a long open channel state induced by large depolarizations. 8. When variable repolarizing potentials were applied after n +80 mV depolarization (5 s), the current‐voltage relationship of the tail current was nearly linear between ‐60 and +30 mV. The deactivation was faster when a larger repolarization step was applied.(ABSTRACT TRUNCATED AT 400 WORDS)
Detection of weak synaptic interactions between single Ia afferent and motor‐unit spike trains in the decerebrate cat.
Authors: Conway B A , Halliday D M , Rosenberg J R
Published: December 2014

1. Spike trains from identified single Ia afferents from soleus and lateral gastrocnemius muscles were recorded (while ‘in continuity’ with the spinal cord) simultaneously with single‐motor‐unit EMG spike trains from the same muscles in decerebrate cats. 2. A total of 143 Ia afferent‐motor‐unit pairs were examined for the presence of correlated activity between the Ia afferent and motor‐unit and between the motor‐unit and Ia afferent. Four types of correlation were identified on the basis of the cross‐intensity function estimated for individual Ia afferent‐motor‐unit pairs. These correlations were attributed to the absence or presence of a central Ia afferent‐motoneurone interaction or a peripheral motor‐unit‐muscle spindle interaction. 3. In addition to the cross‐correlation‐based second‐order cross‐intensity function, third‐order cumulants were defined and used further to investigate Ia afferent‐motor‐unit interactions. A third‐order cumulant density‐based approach to signal processing offers improved signal‐to‐noise ratios, compared with the traditional product density approach, for parameters characterizing certain kinds of linear processes as well as a description of non‐linear interactions. Two classes of third‐order relations were described. One class was associated with a strong central connection and the other with a weak central connection. 4. Third‐order cumulants estimated for Ia afferent‐motor‐unit pairs with significant second‐order central correlations were able to detect a period of decreased motoneuronal excitability. In addition, temporal summation prior to spike initiation could be identified in cases where the afferent discharge was suitably high. 5. Third‐order cumulants estimated for Ia afferent‐motor‐unit pairs in which no significant second‐order central correlation existed identified the presence of weak synaptic interactions. It is argued that these interactions result from the summation from the recorded Ia afferent discharge and other spontaneous synaptic inputs to the motoneurone. 6. The results of the second‐order cross‐intensity analysis of Ia afferent‐motor‐unit interactions, combined with those from the third‐order cumulant density analysis, showed that 77% of the recorded afferents had a detectable influence on motor‐unit behaviour. 7. The results of this study suggest that the third‐order cumulant, based on the analysis of spike trains, will provide a useful tool for detecting synaptic interactions not found by the use of the second‐order cross‐correlation histogram alone, and may also be used to estimate the time course of post‐spike depression in motoneurones, as well as other non‐linear regions of motoneurone membrane trajectory.
The firing rates of human motoneurones voluntarily activated in the absence of muscle afferent feedback.
Authors: Macefield V G , Gandevia S C , Bigland‐Ritchie B , Gorman R B , Burke D
Published: December 2014

1. To quantify the net influence of muscle afferent feedback on the firing rates of human motoneurones, the discharge frequencies of single motor axons in the common peroneal nerve were recorded during sustained voluntary efforts performed in the absence of feedback from the target muscle. These data were compared with the firing rates of single motor units in the intact tibialis anterior muscle. In five subjects, recordings were made from fifty‐two motor axons innervating tibialis anterior during acute deafferentation and paralysis of the dorsiflexor muscles produced by anaesthetic block of the nerve distal to the recording site. 2. Maximal sustainable firing rates were determined for twenty‐four motoneurons, twelve of which were classified as relatively low threshold (estimated recruitment level < or = 10% maximal) and six as high threshold. Mean firing rates of the low‐threshold motoneurones (21.7 +/‐ 2.7 Hz; +/‐ S.E.M.) were significantly higher than those of the high‐threshold motoneurones (14.0 +/‐ 4.4 Hz). The mean firing rate of the twenty‐four deafferented motoneurones during maximal efforts to contract the paralysed muscle was 18.6 +/‐ 1.9 Hz, significantly lower than the maximal firing rates of single motor units recorded from the normally innervated tibialis anterior muscle (28.2 +/‐ 0.6 Hz). 3. During half‐maximal efforts, the mean firing rate of eight deafferented motoneurones (10.8 +/‐ 1.1 Hz) was significantly lower than that of intact motor units (16.5 +/‐ 0.2 Hz). A similar finding was apparent during minimal efforts; the mean discharge frequency of seven deafferented motoneurones during weak voluntary efforts was 6.0 +/‐ 0.9 Hz, compared with 7.3 +/‐ 0.13 Hz for intact motor units. Overall, the range of motoneurone firing rates (from minimal to maximal levels of voluntary effort) was significantly affected by the acute deafferentation, but was shifted significantly to lower rates. 4. During sustained maximal voluntary efforts of at least 30 s duration the firing rate of deafferented motoneurones decreased over the first 5 s but was then maintained, i.e. there was no progressive decline as occurs with normally innervated motor units during fatiguing contractions. This observation supports a reflex origin for the normal decline in motoneurone discharge. 5. It is concluded that muscle afferents in the common peroneal nerve provide a net facilitation to the tibialis anterior motoneurone pool, reflexly increasing the motor output at all levels of voluntary drive by approximately one‐third.
Na+ channels in cardiac and neuronal cells derived from a mouse embryonal carcinoma cell line.
Authors: Arreola J , Spires S , Begenisich T
Published: December 2014

1. Cells from a pluripotent murine embryonal carcinoma cell line (P19) were differentiated in vitro into cells with neurone‐ and cardiac‐like phenotypes. Cells treated with 0.5 microM retinoic acid developed into neurone‐like cells possessing extensive neurites. Dimethyl sulphoxide treatment (0.5%) produced large, spontaneously contracting cell aggregates with many properties of cardiac cells. 2. The neurone‐ and cardiac‐like cells contained voltage‐sensitive Na+ channels with properties similar to those of native neuronal and cardiac cells. 3. We used whole‐cell patch clamp techniques to measure inward currents from the neurone‐ and cardiac‐like cells. Undifferentiated (untreated) cells had only small inward currents (peak of ‐0.15 nA in 150 mM external Na+). The peak inward current in the neurone‐like and cardiac‐like cells was ‐1.2 nA (in 154 mM external Na+) and ‐2.8 nA (in only 46 mM Na+), respectively. These large currents were absent when the external solution contained no Na+. 4. Tetrodotoxin (TTX) blocked the Na+ currents in the neurone‐ and cardiac‐like cells in a dose‐dependent manner. The Kd for TTX block of the Na+ current in the neurone‐like cells was 6.7 nM. The Na+ current in the cardiac‐like cells was much more resistant to TTX; the half‐blocking concentration was two orders of magnitude higher, 710 nM. 5. The kinetic properties of the Na+ channel currents in the neurone‐ and cardiac‐like cells were similar but developed over somewhat different voltage ranges. The voltage sensitivity of activation was similar in both cell types but the activation mid‐point voltage was different: ‐12 mV in the neuronal cells and ‐34 mV for cardiac cells. Inactivation of the neuronal Na+ channels had a mid‐point near ‐47 mV and was more sensitive to the membrane voltage than inactivation of the cardiac channels. The mid‐point of inactivation for the cardiac Na+ channels was ‐80 mV.
Maximum velocity of shortening in relation to myosin isoform composition in single fibres from human skeletal muscles.
Authors: Larsson L , Moss R L
Published: December 2014

1. Maximum velocity of shortening (Vmax) and compositions of myosin heavy chain (MHC) and myosin light chain (MLC) isoforms were determined in single fibres from the soleus or the lateral region of the quadriceps (vastus lateralis) muscles in man. Muscle samples were obtained by percutaneous biopsy, and membranes were permeabilized by glycerol treatment (chemical skinning) or by freeze‐drying. 2. Types I, IIA and IIB MHCs were resolved from single fibre segments by 6% sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and five different fibre types were identified: fibres containing type I MHC, types I and IIA MHCs, type IIA MHC, types IIA and IIB MHCs, and type IIB MHC. Only a few fibres co‐expressed types I and IIA MHCs but 28% of all quadriceps fibres expressed both IIA and IIB MHCs in variable proportions. Fibres co‐expressing types I and IIB MHCs were not found. 3. Alkali (MLC1 and MLC3) and dithio nitrobenzoic acid (DTNB) (MLC2) myosin light chains were observed in all type II fibres in variable proportions. MLC (MLC1s and MLC2s) isoforms from type I fibres had lower migration rates than the corresponding isoforms from type II fibres (MLC1f and MLC2f). More than half of type I fibres in both soleus (65%) and quadriceps (68%) muscles also expressed ‘fast’ MLC3 and 36% of the type II fibres from quadriceps muscle expressed the slow isoform of MLC2. 4. Differences were observed in some mechanical characteristics of freeze‐dried versus chemically skinned fibres. Maximum tension (P0) and specific tension were lower in freeze‐dried types I and IIA fibres than in chemically skinned, while no differences were observed in the IIA/B fibres. The numbers of types I/IIA and IIB fibres were too low to allow statistical comparisons. In chemically skinned fibres, mean specific tension (0.20 +/‐ 0.01 N/mm2) did not vary with fibre type. In freeze‐dried fibres, on the other hand, specific tensions varied according to MHC type: higher (P < 0.01) specific tensions were observed in types IIB (0.19 +/‐ 0.01 N/mm2) and type IIA/B fibres (0.18 +/‐ 0.04 N/mm2) than in type I fibres (0.12 +/‐ 0.02 N/mm2). The specific tension of type IIA fibres (0.12 +/‐ 0.05 N/mm2) did not differ significantly from the other fibre types. Cross‐sectional areas and mean Vmax did not differ between freeze‐dried and chemically skinned fibres, either when all fibres were pooled or within respective fibre types. Vmax data from all fibres of a given type, irrespective of membrane permeabilization technique, have therefore been pooled.(ABSTRACT TRUNCATED AT 400 WORDS)
Quantal components of unitary EPSCs at the mossy fibre synapse on CA3 pyramidal cells of rat hippocampus.
Authors: Jonas P , Major G , Sakmann B
Published: December 2014

1. Excitatory postsynaptic currents (EPSCs) were recorded in CA3 pyramidal cells of hippocampal slices of 15‐ to 24‐day‐old rats (22 degrees C) using the whole‐cell configuration of the patch clamp technique. 2. Composite EPSCs were evoked by extracellular stimulation of the mossy fibre tract. Using the selective blockers 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) and D‐2‐amino‐5‐phosphonopentanoic acid (APV), a major alpha‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (AMPA)/kainate receptor‐mediated component and a minor NMDA receptor‐mediated component with slower time course were distinguished. For the AMPA/kainate receptor‐mediated component, the peak current‐voltage (I‐V) relation was linear, with a reversal potential close to 0 mV. The half‐maximal blocking concentration of CNQX was 353 nM. 3. Unitary EPSCs of the mossy fibre terminal (MF)‐CA3 pyramidal cell synapse were evoked at membrane potentials of ‐70 to ‐90 mV by low‐intensity extracellular stimulation of granule cell somata using fine‐tipped pipettes. The EPSC peak amplitude as a function of stimulus intensity showed all‐or‐none behaviour. The region of low threshold was restricted to a few micrometres. This suggests that extracellular stimulation was focal, and that the stimulus‐evoked EPSCs were unitary. 4. Latency and rise time histograms of EPSCs evoked by granule cell stimulation showed narrow unimodal distributions within each experiment. The mean latency was 4.2 +/‐ 1.0 ms, and the mean 20‐80% rise time was 0.6 +/‐ 0.1 ms (23 cells). When fitted within the range 0.7 ms to 20 ms after the peak, the decay of the EPSCs with the fastest rise (rise time 0.5 ms or less) could be described by a single exponential function; the mean time constant was in the range 3.0‐6.6 ms with a mean of 4.8 ms (8 cells). 5. Peak amplitudes of the EPSCs evoked by suprathreshold granule cell stimulation fluctuated between trials. The apparent EPSC peak conductance in normal extracellular solution (2 mM Ca2+, 1 mM Mg2+), excluding failures, was 1 nS. Reducing the Ca2+ concentration and increasing the Mg2+ concentration reduced the mean peak amplitude in a concentration‐dependent manner. 6. Peaks in EPSC peak amplitude distributions were apparent in low Ca2+ and high Mg2+. Using the criteria of equidistance and the presence of peaks and dips in the autocorrelation function, five of nine EPSC peak amplitude distributions were judged to be quantal.(ABSTRACT TRUNCATED AT 400 WORDS)
Exocytosis elicited by action potentials and voltage‐clamp calcium currents in individual mouse pancreatic B‐cells.
Authors: Ammälä C , Eliasson L , Bokvist K , Larsson O , Ashcroft F M , Rorsman P
Published: December 2014

1. Measurements of membrane capacitance, as an indicator of exocytosis, and intracellular Ca2+ concentration ([Ca2+]i) were used to determine the Ca2+ dependence of secretion in single pancreatic B‐cells. 2. Exocytosis was dependent on a rise in [Ca2+]i and could be evoked by activation of voltage‐dependent Ca2+ currents. The threshold for depolarization‐induced release was 0.5 microM [Ca2+]i. Once the [Ca2+]i threshold was exceeded, exocytosis was rapidly (< 50 ms) initiated. When individual pulses were applied, exocytosis stopped immediately upon repolarization and the Ca2+ channels closed, although [Ca2+]i remained elevated for several seconds. 3. During repetitive stimulation (1 Hz), when [Ca2+]i attained micromolar levels, exocytosis also took place during the interpulse intervals albeit at a slower rate than during the depolarizations. 4. Exocytosis could be initiated by simulated action potentials. Whereas a single action potential only produced a small capacitance increase, and in some cells even failed to stimulate release, larger and more consistent responses were obtained with > or = four action potentials. 5. Comparison of the rates of exocytosis measured in response to depolarization, mobilization of Ca2+ from intracellular stores or infusion of Ca2+ through the patch pipette suggests that [Ca2+]i at the secretory sites attains a concentration of several micromolar. This is much higher than the average [Ca2+]i detected by microfluorimetry suggesting the existence of steep spatial gradients of [Ca2+]i within the B‐cell. 6. Inclusion of inhibitors of Ca2+/calmodulin‐dependent protein kinase II in the intracellular solution reduced the depolarization‐induced exocytotic responses suggesting this enzyme may be involved in the coupling between elevation of [Ca2+]i to stimulation of the secretory machinery. 7. The size of the unitary exocytotic event was 2 fF, corresponding to a secretory granule diameter of 250 nm. 8. Over short periods, exocytosis may be extremely fast (1 pF/s or 500 granules/s), which is much higher than the rate of endocytosis (18 fF/s or 9 granules/s). Since the latter is in better agreement with the maximum rate of insulin secretion from islets (approximately 2 granules/s), we suggest that membrane retrieval may set an upper limit on the rate of exocytosis during extended periods of secretion.
Mechanical determinants of gradient walking energetics in man.
Authors: Minetti A E , Ardigò L P , Saibene F
Published: December 2014

1. The metabolic cost and the mechanical work at different speeds during uphill, level and downhill walking have been measured in four subjects. 2. The mechanical work has been partitioned into the internal work (W(int)), due to the speed changes of body segment with respect to the body centre of mass (BCM), and the external work (W(ext)), related to the position and speed changes of the BCM in the environment. 3. W(ext) has been further divided into a positive part W+ext) and a negative one (W‐(ext)), associated with the energy increases and decreases, respectively, over the stride period. 4. For all constant speeds the most economical gradient has been found to be ‐10.2% (+/‐ 0.8 S.D.). 5. At each gradient there is a unique W+ext/W‐ext ratio (= 1 in level walking), regardless of speed, with a tendency for W‐ext and W+ext to vanish above +15% and below ‐15% gradient, respectively. 6. W(int) is constant at each speed regardless of gradient. This is partly explained by an only slight decrease in stride frequency at increasing gradient. W(int) constancy implies that it has no role in determining the optimum gradient. 7. A linear multiple regression relating W+ext and W‐ext to the metabolic cost at different gradients showed that negative (eff‐) and positive (eff+) efficiencies decrease with increasing speed (from 0.912 to 0.726, and from 0.182 to 0.146, respectively). The eff‐/eff+ ratio, however, remains rather constant (4.995 +/‐ 0.125 S.D.). 8. We conclude that the measured W(ext), the W+ext/W‐ext partitioning and eff‐/eff+ ratio, i.e. the different efficiency of the muscles used as force and brake generators, can explain the metabolic optimum gradient at about ‐10%.
Muscarine modulation by a G‐protein alpha‐subunit of delayed rectifier K+ current in rat ventromedial hypothalamic neurones.
Authors: ffrench‐Mullen J M , Plata‐Salamán C R , Buckley N J , Danks P
Published: December 2014

1. Rat cultured ventromedial hypothalamic (VMH) neurones obtained from embryonic hypothalamus were used to study the muscarinic (carbachol) modulation of voltage‐gated K+ currents with the whole‐cell patch‐clamp technique. 2. Carbachol produced a potent and concentration‐dependent (100 fM to 100 microM) decrease of the outward delayed rectifier K+ current (IK) with an IC50 of 44 pM and a Hill coefficient of 0.4. The carbachol‐induced depression of IK was reduced by pirenzepine (1‐10 microM) and atropine (1 microM). Carbachol had no effect on the transient outward K+ current (IA). 3. Intracellular dialysis with guanosine 5'‐O‐(2‐thiodiophosphate) (GDP‐beta‐S, 500 microM) significantly diminished the carbachol‐induced depression of IK, suggesting GTP‐binding protein (G‐protein) involvement. Pre‐incubation of VMH neurones with pertussis toxin (200‐400 ng ml‐1) or cholera toxin (1 microgram ml‐1) for 24‐48 h had no effect on the carbachol‐induced depression of IK. This suggested that the G alpha o, G alpha i, and G alpha s G‐protein alpha‐subunits were not involved in mediating the carbachol‐induced depression of IK in VMH neurones. 4. Treatment (24‐48 h) of VMH neurones with antisense phosphothio‐oligodeoxynucleotides to the G alpha 11 G‐protein subunit (10 microM) significantly diminished the carbachol‐induced depression of IK. Treatment with 10 microM of either G alpha 11 sense or antisense to G alpha q had no effect. 5. These results demonstrate a novel and potent muscarinic depression of IK in VMN neurones, and that this depression is specifically mediated by the G alpha 11 G‐protein subunit.(ABSTRACT TRUNCATED AT 250 WORDS)
A longitudinal gradient of synaptic drive in the spinal cord of Xenopus embryos and its role in co‐ordination of swimming.
Authors: Tunstall M J , Roberts A
Published: December 2014

1. Intersegmental co‐ordination in Xenopus embryos could be influenced by longitudinal gradients in neuronal properties or synaptic drive. To determine if such gradients exist intracellular recordings were made from putative motoneurones at different spinal levels. 2. No evidence was found of a longitudinal gradient in neuronal resting potentials. In a rostrocaudal direction the duration of current‐evoked spikes increased and the amplitude of the spike after‐hyperpolarization (AHP) decreased. 3. During fictive swimming the amplitude of the tonic excitatory synaptic input and the mid‐cycle IPSPs declined in a rostrocaudal direction. The rise‐time and fall‐time of mid‐cycle IPSPs increased in a rostrocaudal direction. 4. Rostral to the eighth post‐otic segment mid‐cycle IPSPs occurred on all cycles of fictive swimming episodes. More caudally IPSPs became irregular in occurrence and caudal to the twelfth post‐otic segment no mid‐cycle IPSPs could be detected, even during the injection of depolarizing current or when recording with KCl‐filled electrodes. 5. The duration of spikes occurring during fictive swimming increased and the amplitude of spike AHP decreased in a rostrocaudal direction. A spike AHP was absent during fictive swimming activity in neurones caudal to the ninth post‐otic segment even though it was present in current‐evoked spikes in the same neurones. 6. On‐cycle IPSPs (occurring shortly after the spike at phase values less than 0.4) were observed predominantly at the beginning of swimming episodes in neurones recorded rostral to the eighth segment, but were not detected at all in more caudal neurones. 7. If the rostrocaudal gradients in synaptic excitatory and inhibitory drive to putative motoneurones during fictive swimming are also present in premotor spinal interneurones they would be expected to have a strong influence on rostrocaudal delays. Such gradients could therefore be important components of the mechanism underlying intersegmental co‐ordination.
Calcitonin gene‐related peptide activated ATP‐sensitive K+ currents in rabbit arterial smooth muscle via protein kinase A.
Authors: Quayle J M , Bonev A D , Brayden J E , Nelson M T
Published: December 2014

1. Whole‐cell K+ currents activated by calcitonin gene‐related peptide (CGRP) in smooth muscle cells enzymatically isolated from rabbit mesenteric arteries were measured in the conventional and perforated configurations of the patch clamp technique. The signal transduction pathway from CGRP receptors to activation of potassium currents was investigated. 2. CGRP (10 nM) activated a whole‐cell current that was blocked by glibenclamide (10 microM), an inhibitor of ATP‐sensitive K+ channels. Elevating intracellular ATP reduced glibenclamide‐sensitive currents. CGRP increased the glibenclamide‐sensitive currents by 3‐ to 6‐fold in cells dialysed with 0.1 mM ATP, 3.0 mM ATP or in intact cells. The reversal potential of the glibenclamide‐sensitive current in the presence of CGRP shifted with the potassium equilibrium potential, while its current‐voltage relationship exhibited little voltage dependence. 3. Forskolin (10 microM), an adenylyl cyclase activator, Sp‐cAMPS (500 microM) and the catalytic subunit of protein kinase A increased glibenclamide‐sensitive K+ currents 2.1‐, 3.3‐ and 8.2‐fold, respectively. 4. Nitric oxide and nitroprusside did not activate glibenclamide‐sensitive K+ currents. 5. Dialysis of the cell's interior with inhibitors of protein kinase A (synthetic peptide inhibitor, 4.6 microM or H‐8, 100 microM) completely blocked activation of K+ currents by CGRP. 6. Our results suggest the following signal transduction scheme for activation of K+ currents by CGRP in arterial smooth muscle: (1) CGRP stimulates adenylyl cyclase, which leads to an elevation of cAMP; (2) cAMP activates protein kinase A, which opens ATP‐sensitive K+ channels.