bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanism of release of Ca2+ from intracellular stores in response to ionomycin in oocytes of the frog Xenopus laevis.
Authors: Yoshida S , Plant S
Published: December 2014

1. The mechanism of Ca2+ release from intracellular stores was studied in defolliculated Xenopus laevis oocytes by measuring whole‐cell currents using the two‐electrode voltage‐clamp method. 2. The extracellular application of ionomycin, a selective Ca2+ ionophore, evoked an inward current consisting of a spike‐like fast component followed by a long‐lasting slow component with few superimposed current oscillations (fluctuations). The ionomycin response occurred in a dose‐dependent manner and was dependent on Cl‐. 3. No apparent refractory period was observed for repetitively evoked small ionomycin responses when the concentration of ionomycin was low (0.1 microM). In contrast, a larger ionomycin response (1 microM), consisting of fast and slow components, was followed by refractory period. Washing for 50‐90 min was necessary for full recovery of the ionomycin response. 4. The response to ionomycin was suppressed by the extracellular application of acetoxymethyl ester of bis‐(O‐aminophenoxy)‐ethane‐N,N,N',N'‐tetraacetic acid (BAPTA AM, 1‐10 microM), a membrane‐permeable intracellular Ca2+ chelator. 5. The ionomycin response was not affected by pertussis toxin (PTX, 0.3‐2.0 microgram/ml), a blocker of guanine nucleotide‐binding regulatory proteins (G proteins). In contrast, the response to acetylcholine (ACh), which is known to occur via a G protein, was suppressed by PTX. 6. The fast component was not affected by removing Ca2+ from the bathing medium or by replacing extracellular Ca2+ with Ba2+ or Mn2+ (all of these solutions were supplemented with 2 mM EGTA), whereas the slow component was suppressed. 7. Injection of inositol 1,4,5‐trisphosphate (IP3) following a response to extra‐cellularly applied ionomycin did not evoke an appreciable membrane current. In contrast, ionomycin evoked a small inward current when it was applied after an inward‐current response evoked by IP3 injection, whereas a second injection of IP3 did not evoke any appreciable current. 8. The results indicate that (a) ionomycin releases Ca2+ from its intracellular stores without the involvement of G proteins, resulting in activation of Ca(2+)‐activated Cl‐ channels, (b) ionomycin mainly acts on the same intracellular Ca2+ stores as IP3, and (c) entry of Ca2+ from outside the cell considerably contributes to the slow component of the ionomycin response, whereas its fast component is predominantly dependent on the release of Ca2+ from the intracellular stores.
Magnetic coil stimulation of straight and bent amphibian and mammalian peripheral nerve in vitro: locus of excitation.
Authors: Maccabee P J , Amassian V E , Eberle L P , Cracco R Q
Published: December 2014

1. According to classical cable theory, a magnetic coil (MC) should excite a linear nerve fibre in a homogeneous medium at the negative‐going first spatial derivative of the induced electric field. This prediction was tested by MC stimulation of mammalian phrenic and amphibian sciatic nerve and branches in vitro, immersed in Ringer solution within a trough, and identifying the sites of excitation by recording responses of similar latency to local electrical stimulation. Subsequently, the identified sites of excitation were compared with measurements of the induced electric field and its calculated first spatial derivative. A special hardware device was used to selectively reverse MC current direction and to generate predominantly monophasic‐ or polyphasic‐induced pulse profiles whose initial phases were identical in polarity, shape and amplitude. When using the amphibian nerve preparation, a complication was excitation at low threshold points related to cut branches. 2. Reversal of monophasic current resulted in latency shifts corresponding approximately to the distance between induced cathode and anode. The location of each site of excitation was at, or very near, the negative‐going first spatial derivative peaks of the induced electric field measured parallel to the straight nerve. Significantly, excitation of the nerve did not occur at the peak of the induced electric field above the centre of the ‘figure of eight’ MC junction. 3. A polyphasic pulse excited the nerve at both sites, by the negative‐going first phase at one location, and approximately 150 microseconds later, by the reversed negative‐going second phase at the other location. Polyphasic and monophasic pulses elicited responses with similar latency when the induced current flowed towards the recording electrode. 4. Straddling a nerve with non‐coding solid lucite cylinders created a localized spatial narrowing and increase in the induced electric field, resulting in a lowered threshold of excitation. The corresponding closer spacing between first spatial derivative peaks was exhibited by a significant reduction in latency shift when MC current direction was reversed. 5. When a nerve is bent and the induced current is directed along the nerve towards the bend, the threshold of excitation is reduced there. Increasing the angle of the bend from 0 deg to more than 90 deg graded the decrease in threshold. 6. In a straight nerve the threshold was lowest when current was directed towards the cut end.(ABSTRACT TRUNCATED AT 400 WORDS)
Impairment of neuromuscular propagation during human fatiguing contractions at submaximal forces.
Authors: Fuglevand A J , Zackowski K M , Huey K A , Enoka R M
Published: December 2014

1. The purpose of the study was to examine the dependence of neuromuscular propagation impairment on the level of isometric force sustained to the endurance limit. The task involved human volunteers sustaining a submaximal abduction force with the index finger by activating the first dorsal interosseous muscle as long as possible. 2. The submaximal force was sustained at one of three levels (20, 35 or 65% of maximum) by increasing motor unit activity, as indicated by the electromyogram (EMG), during the fatiguing contraction. Although the EMG increased during the fatiguing contraction, the EMG was significantly less than maximum at the endurance limit for all subjects (deficit of 19‐55% of maximum). This deficit was inversely related to the level of the sustained submaximal force. 3. The maximum voluntary contraction and twitch forces were significantly reduced following the fatiguing contraction. As with the EMG, the degree of force reduction was greatest for the subjects who sustained the low target forces. 4. The fatiguing contraction caused a 12‐23% decline in M wave amplitude, a 33‐51% increase in M wave duration, and no change in M wave area. The decline in M wave amplitude, which is an index of neuromuscular propagation impairment, was greatest among the subjects who sustained the low target forces. 5. The mean power frequency of the EMG decreased by a similar amount (50‐57%) during the fatiguing contraction for all three groups of subjects. 6. A model representing the interaction of processes that enhance and impair force was developed to explain the recovery of twitch force following the sustained contractions at different target forces. 7. We conclude that the fatigue experienced by a subject when force is sustained at a submaximal value does involve an impairment of neuromuscular propagation. This impairment is one factor that limits muscle excitation during a submaximal, fatiguing contraction and contributes to the diminished force capability by the end of the fatigue task.
Modulation of respiratory activity of neonatal rat phrenic motoneurones by serotonin.
Authors: Lindsay A D , Feldman J L
Published: December 2014

1. The effects of serotonin on phrenic motoneurones were studied in an in vitro preparation of the isolated brainstem and spinal cord from neonatal rats. 2. Serotonin (5‐HT; > or = 5‐10 microM) increased inspiratory‐modulated phrenic nerve activity and produced a small amount of tonic activity during expiration. Inspiratory‐modulated activity of the fourth cervical ventral root also increased, but was accompanied by robust tonic activity, which often obscured the rhythmic activity. 3. Serotonin, in both normal and tetrodotoxin‐containing medium, depolarized phrenic motoneurones and increased cell input resistance. Serotonin also increased inspriatory‐modulated firing as well as the response of phrenic motoneurones to injected current. The y‐intercept of the relationship between firing frequency and injected current (f‐I) was increased, but the slope was not affected. There was no bistable firing behaviour. 4. Under voltage clamp conditions, 5‐HT produced a tonic inward current of 0.07‐0.37 nA. This current increased with less negative holding potentials and decreased with more negative holding potentials (‐75 to ‐90 mV) but did not reverse. 5. In addition, 5‐HT decreased inspiratory‐modulated synaptic current by 23 +/‐ 6%. The degree of attenuation was not affected by holding potential. The time course of the decrease in inspiratory‐modulated synaptic current was similar to the changes seen in tonic inward current and input resistance. 6. Depolarization, tonic inward current, and shift in the f‐I relationship produced by 5‐HT were antagonized by the 5‐HT2/1C receptor antagonist ketanserin and mimicked by the 5‐HT2/1C agonist 1‐(2,5‐dimethoxy‐4‐iodophenyl)‐2‐aminopropane HCl (DOI). However, the 5‐HT induced decrease in inspiratory‐modulated synaptic current was not reduced by ketanserin nor mimicked by DOI. 7. We conclude that exogenously applied 5‐HT simultaneously increases cell excitability and decreases inspiratory‐modulated synaptic current in phrenic motoneurones via different types of receptors. When these responses occurred simultaneously, the increase in excitability predominated and the net effect was an augmentation of inspiratory‐modulated phrenic motoneurone activity.
A statistical analysis of acetylcholine receptor activation in Xenopus myocytes: stepwise versus concerted models of gating.
Authors: Auerbach A
Published: December 2014

1. The kinetic properties of single channel currents from fetal‐type acetylcholine receptors in embryonic Xenopus myocytes (60 h old) have been analysed by a maximum‐likelihood method. 2. At very high acetylcholine (ACh) concentrations (up to 5 mM) the effective opening rate appears to saturate at approximately 30,000 s‐1. 3. The kinetics were analysed according to the standard concerted scheme that postulates a single channel‐opening conformational change after two agonists are bound, and a rarely invoked stepwise scheme that postulates semi‐independent conformational changes in two distinct gating domains. Both models assume that agonist cannot escape from a channel (or domain) that is in its activated conformation. 4. With either activation scheme the kinetic analyses indicate that ACh binds at a rate of approximately 2 x 10(8) s‐1 M‐1 and dissociates from doubly liganded receptors at a rate of approximately 28,000 s‐1, and that the activation process is asymmetric, i.e. the binding (concerted model) or gating (stepwise model) transitions are not equal and independent. 5. In eighteen of twenty‐seven file‐by‐file comparisons, the likelihood of the stepwise model was greater than that of the concerted model. In seven such comparisons, the likelihood of the concerted model was greater than that of the stepwise model, and in two there was no difference. Log likelihood ratio distributions were obtained from three files (those with the most events) by multiple cycles of resampling and fitting. The means of these distributions were significantly greater than zero, indicating that the stepwise scheme was as good as, or better than, the concerted scheme in describing receptor activation. 6. According to the stepwise view, two binding sites must be occupied and two ‘gates’ activated for conduction to occur. Although equivalent binding is not an essential aspect of stepwise activation, the binding sites can be identical and have a low affinity for ACh (Kd approximately 130 microM). Either the isomerization rates of the gating domains are different, or they are influenced by the conformational status of its counterpart, with activation increasing approximately 3‐fold and deactivation decreasing approximately 10‐fold if the complementary domain is in the active conformation. Stepwise activation predicts that the decay of the endplate current is determined by five rates.
Contralateral projections of thoracic respiratory interneurones in the cat.
Authors: Schmid K , Kirkwood P A , Munson J B , Shen E , Sears T A
Published: December 2014

1. Retrograde neuronal transport following small iontophoretic injections of horseradish peroxidase was used to investigate the location of neurones projecting to the thoracic ventral horn of the cat. 2. A concentration of labelled neurones was seen in the contralateral medial ventral horn at the immediately opposite rostrocaudal position. 3. The participation of respiratory interneurones in the projection was investigated by means of spike‐triggered averaging, as follows. 4. Spike trains of single interneurones whose firing patterns were related to the central respiratory cycle were recorded extracellularly in the thoracic ventral horn of anaesthetized, paralysed cats. Firing patterns were defined by the construction of cycle‐triggered histograms. 5. Spike‐triggered averaging of the signal from an extracellular tungsten microelectrode in the opposite ventral horn was performed to test for the presence of axonal, terminal or focal synaptic potentials. 6. At least one of these types of potential was found for 34/55 units. Terminal potentials were found for thirty‐one units, accompanied by focal synaptic potentials for twenty‐seven units. Potentials were found for units with all types of firing patterns. Units whose activity elicited these potentials were generally located in the medial half of the ventral horn. 7. We conclude that at least 60% of the respiratory interneurones project to the immediately opposite ventral horn.
Changes in the discharge patterns of cat motor cortex neurones during unexpected perturbations of on‐going locomotion.
Authors: Marple‐Horvat D E , Amos A J , Armstrong D M , Criado J M
Published: December 2014

1. The impulse activity of single neurones in the forelimb part of the motor cortex was recorded extracellularly in unrestrained cats during self‐paced locomotion on a horizontal circular ladder. 2. Fifty‐one cells (forty‐nine of which discharged rhythmically in time with the step cycle) were recorded during encounters with a number of rungs that could be locked firmly in position or, alternatively, held in position by weak springs so that when stepped on they unexpectedly descended (under the weight of the animal) from 1 to 5 cm before contacting a mechanical stop. 3. In eleven cells (22%) including four fast‐axon pyramidal tract neurones (PTNs), an increase in discharge occurred when the contralateral forelimb descended unexpectedly. Onset latency relative to the start of rung movement ranged from ca 20 to ca 100 ms. In eight cells latency was such that most of the response preceded contact of the rung with the stop; averaged over a number of trials the altered discharge in five of these cells (including two PTNs) represented an accurate profile of the averaged velocity of rung (and foot) descent. The three remaining cells appeared to be responding largely to the cessation of rung movement. 4. Thirty‐six of the cells were also studied during unexpected descent of the ipsilateral forelimb and six (17%) displayed an increase in discharge (onset latency ca 35 to ca 80 ms); three of these were among those that also responded to contralateral descents. 5. These findings for skilled locomotion requiring a high degree of visuomotor coordination are discussed and it is concluded that the motor cortex is rapidly informed regarding unexpected perturbations delivered to the contralateral forelimb at the onset of stance and that changes are evoked in the pattern of impulse traffic descending via the pyramidal tract.
Influence of force on muscle and skin sympathetic nerve activity during sustained isometric contractions in humans.
Authors: Seals D R
Published: December 2014

1. Our purpose was to test the hypothesis that efferent sympathetic nerve activity to non‐active skeletal muscle (MSNA) and skin (SSNA) is independent of the level of force during sustained submaximal isometric contractions in humans. 2. In twelve healthy subjects, arterial blood pressure, heart rate, and MSNA (n = 6) or SSNA (n = 6) (peroneal microneurography) were recorded before and during isometric handgrip contractions sustained to exhaustion at 20, 40 and 60% of maximal force. Responses were examined at similar percentages of endurance time at each level of force. 3. Contraction duration decreased progressively with increasing force (495 +/‐ 54, 140 +/‐ 13, 73 +/‐ 8 s, respectively), but peak ratings of perceived effort were similar for the three force levels. 4. The peak increases in systolic pressure were not different among the three levels of force. The increases in diastolic and mean pressure were similar at 40 and 60% of maximal force, but were smaller at the end of 20% of maximal force. The contraction‐induced rise in heart rate was directly related to the level of force. 5. The contraction‐evoked stimulation of both MSNA and SSNA was similar during handgrip at 40 and 60% of maximal force, but was much less during handgrip at 20% of maximal force. The increases in SSNA were associated with increases in both skin blood flow and skin electrical conductance suggesting primarily sudomotor fibre activation. 6. These findings indicate that there is a minimum force necessary to elicit peak levels of MSNA and SSNA during sustained isometric contractions in humans. When normalized to endurance time, however, the regulation of these sympathetic outflows appears to be independent of force above this minimum level. The results also indicate that during this type of muscle activity the relationship between force and heart rate is different to that between force and peripheral sympathetic discharge.
Influence of stimulation parameters on the release of adenosine, lactate and CO2 from contracting dog gracilis muscle.
Authors: Achike F I , Ballard H J
Published: December 2014

1. The addition of adenosine, CO2 and lactate to the venous blood draining an isolated constant‐flow perfused gracilis muscle was studied in anaesthetized and artificially ventilated dogs during twitch and tetanic contractions. 2. Venous adenosine concentration increased from 154 +/‐ 33 nM (mean +/‐ S.E.M.) to 279 +/‐ 121 or 280 +/‐ 125 nM after 10 min of 1.5 or 3 Hz twitch contractions and to 240 +/‐ 120 or 276 +/‐ 139 nM after 10 min of 1 or 5 s tetani occurring at 0.1 Hz. Twitch contractions at 0.1 or 0.5 Hz for 10 min did not significantly elevate venous adenosine. 3. Venous lactate concentration was significantly increased after 10 min of 1.5 or 3 Hz twitches or 5 s tetani at 0.1 Hz. There was a good correlation (r = 0.70; P < 0.001) between venous adenosine and lactate concentrations. 4. Venous partial pressure of CO2 (PCO2) was significantly elevated after 10 min of 1.5 or 3 Hz twitch contractions or 1 or 5 s tetani at 0.1 Hz. There was also a good correlation (r = 0.58; P < 0.001) between venous adenosine concentration and PCO2. 5. Venous partial pressure of O2 (PO2) decreased during all contractions except those at 0.1 Hz, but the oxygen cost per unit of tension x time was similar during every pattern of stimulation, and the percentage of the total energy production achieved by anaerobic means during muscle contractions did not exceed that at rest, indicating that there had been no limitation to the oxygen supply. Venous PO2 was poorly correlated with venous adenosine concentration (r = 0.28), but quite well correlated with venous lactate concentration (r = 0.53; P < 0.001). If the indirect influence of PO2 on venous adenosine concentration via an increase in lactate concentration was eliminated by partial correlation, then the coefficient for the relationship between venous adenosine concentration and venous PO2 became 0.15. 6. There was a significant correlation between the venous adenosine concentration and the venous pH (r = 0.53; P < 0.001). If the influence of oxygenation on venous adenosine and pH was eliminated by partial correlation, the coefficient for the relationship between venous adenosine and pH increased to 0.95.(ABSTRACT TRUNCATED AT 400 WORDS)
Calcium transients in single mammalian skeletal muscle fibres.
Authors: Delbono O , Stefani E
Published: December 2014

1. We studied the transient changes in myoplasmic Ca2+ concentration under current‐ and voltage‐clamp (double Vaseline‐gap technique) in cut fibres of rat extensor digitorum longus muscle using mag‐fura‐2 (furaptra) as Ca2+ indicator, at 3.6‐3.8 microns sarcomere length and 17 degrees C. Mag‐fura‐5 and fura‐2 were also used in order to characterize some aspects of the Ca2+ transients. 2. The peak [Ca2+] in response to a single action potential was 4.6 +/‐ 0.4 microM (n = 5). The time to peak of the Ca2+ transient was 4.6 +/‐ 0.42 ms, with half‐width of 8.2 +/‐ 1.5 ms, time constant of the rising phase 1.15 +/‐ 0.25 ms, time constant of the decaying phase 3.26 +/‐ 0.65 ms, and delay between action potential and Ca2+ transient 2.0 +/‐ 0.2 ms. 3. Ca2+ transients were studied under voltage‐clamp conditions at different voltages and pulse durations. The rising phase showed a complex temporal course with a fast initial increase and a second component. Both components were separated by a plateau or a brief decrease of the Ca2+ concentration. The peak Ca2+ transient was 10.5 +/‐ 1.3 microM (n = 22). 4. After interrupting the pulse, Ca2+ concentration decayed exponentially. The time constant of decay of the Ca2+ transient increased with the pulse voltage and duration, reaching a maximum value at potentials more positive than +10 mV and pulses longer than 200 ms. An analysis of the decaying phases of the Ca2+ transients suggests that only the removal process operates after fibre repolarization. 5. The rate of Ca2+ release from the sarcoplasmic reticulum was calculated using the Melzer, Ríos & Schneider model. The value of 17.2 +/‐ 3.1 micronM ms‐1 (n = 10) estimated in these calculations was intermediate between those obtained by other authors from cut frog muscles (10 microM ms‐1) and intact frog fibres (100 microM ms‐1) using antipyrylazo III (AP III) as the Ca2+ indicator.
Multiple mechanisms of picrotoxin block of GABA‐induced currents in rat hippocampal neurons.
Authors: Yoon K W , Covey D F , Rothman S M
Published: December 2014

1. We have examined the effect of picrotoxin on GABA‐induced currents in dissociated rat hippocampal neurons. In addition, we used the putative picrotoxin receptor antagonist, alpha‐isopropyl‐alpha‐methyl‐gamma‐butyrolactone (alpha IMGBL), and the picrotoxin agonist, beta‐ethyl‐beta‐methyl‐gamma‐butyrolactone (beta EMGBL) to explore the mechanisms of picrotoxin's interaction with the GABA‐Cl‐ receptor‐ionophore complex. 2. The picrotoxin block of GABA current was use dependent, suggesting that the site of picrotoxin block is exposed by the conformational change initiated by GABA binding to the receptor. 3. The alkyl‐substituted butyrolactone antagonist, alpha IMGBL, selectively blocked the use‐dependent mechanism of picrotoxin effect. After the apparent complete inhibition of the use‐dependent effect, there was a residual picrotoxin effect that was independent of the time or concentration of GABA application. This indicates that the picrotoxin block of the GABA current is mediated by two different mechanisms. alpha IMGBL influences just one of these mechanisms. 4. The picrotoxin receptor agonist, beta EMGBL, exclusively blocked the GABA current in a use‐dependent manner. Consistent with a use‐dependent mechanism, the rate of onset of block increased with GABA concentration. Surprisingly, the fraction of GABA current block decreased with increasing GABA concentration. 5. These results suggest that the relationship of picrotoxin and gamma‐butyrolactones with the GABA‐Cl‐ receptor‐ionophore is quite complex. They are consistent with at least two possible models of agonist‐antagonist interactions. Both cases require different antagonist affinities for the various kinetic states of the GABA‐Cl‐ receptor‐ionophore. However, there is no need to require that either picrotoxin or beta EMGBL acts as an open channel blocker.
On the role of G protein activation and phosphorylation in desensitization to acetylcholine in guinea‐pig atrial cells.
Authors: Zang W J , Yu X J , Honjo H , Kirby M S , Boyett M R
Published: December 2014

1. The ACh‐activated K+ current (IK,ACh) has been investigated in guinea‐pig atrial cells at 36 degrees C using the whole‐cell patch‐clamp technique. 2. During an exposure to ACh, IK,ACh faded as a result of desensitization. Throughout the fade of the current, the current reversed at EK and showed inward‐going rectification. The fade was, therefore, the result of a genuine decrease in IK,ACh. 3. The onset of desensitization (as judged by the fade of IK,ACh) was biphasic and the time constants of the fast and slow phases of desensitization were 1.58 +/‐ 0.14 (n = 16) and 148.2 +/‐ 12.8 s (n = 18) respectively. Recovery from the fast and slow phases of desensitization (after 30 s and 5 min exposures to ACh respectively) occurred with time constants of 52 and 222 s respectively. This suggests that two processes are involved in desensitization. 4. The Q10 of the rate constant of the fast phase of desensitization was 2.2 +/‐ 0.3 (n = 6). 5. Intracellular perfusion with guanosine 5'‐O‐(3‐thiotriphosphate) (GTP gamma S) or extracellular perfusion with AlF4‐ were used to bypass the muscarinic receptor and trigger IK,ACh by directly activating the G protein, GK, that links the muscarinic receptor to the K+ channel. Both GTP gamma S and AlF4‐ activated a current with the same reversal potential and the same degree of inward‐going rectification as the ACh‐activated current. 6. Desensitization still occurred when the muscarinic receptor was bypassed and IK,ACh was triggered by direct activation of GK with either GTP gamma S or AlF4‐. This suggests that desensitization is, in part, the result of a modification of either GK or the K+ channel. 7. Activation of the muscarinic receptor by ACh resulted in greater desensitization than direct activation of GK; at the end of a 5 min exposure to ACh, current was only 22 +/‐ 1% (n = 19) of its peak value, whereas, after direct activation of GK by GTP gamma S for 5 min, current was 42 +/‐ 6% (n = 5) of its peak value. This suggests that desensitization also involves the muscarinic receptor. 8. When cells were perfused with GTP gamma S, the fast phase of desensitization could still occur, but the slow phase was reduced. This suggests that the fast phase involves GK or the K+ channel, whereas the slow phase involves the muscarinic receptor.(ABSTRACT TRUNCATED AT 400 WORDS)
Pulmonary stretch receptor afferents activate excitatory amino acid receptors in the nucleus tractus solitarii in rats.
Authors: Bonham A C , Coles S K , McCrimmon D R
Published: December 2014

1. The goal of the present study was to identify potential neurotransmitter candidates in the Breuer‐Hering (BH) reflex pathway, specifically at synapses between the primary afferents and probable second‐order neurones (pump cells) within the nucleus tractus solitarii (NTS). We hypothesized that if activation of specific receptors in the NTS is required for production of the BH reflex, then (1) injection of the receptor agonist(s) would mimic the reflex response (apnoea), (2) injection of appropriate antagonists would impair the apnoea produced by either lung inflation or agonist injection, and (3) second‐order neurones in the pathway would be excited by either lung inflation or agonists while antagonists would prevent the response to either. 2. Studies were carried out either in spontaneously breathing or in paralysed, thoracotomized and ventilated rats in which either diaphragm EMG or phrenic nerve activity, expired CO2 concentration and arterial pressure were continuously monitored. The BH reflex was physiologically activated by inflating the lungs. 3. Pressure injections (0.03‐15 pmol) of selective excitatory amino acid (EAA) receptor agonists, quisqualic acid (Quis) and N‐methyl‐D‐aspartic acid (NMDA) into an area of the NTS shown previously to contain neurones required for production of the BH reflex produced dose‐dependent apnoeas that mimicked the response to lung inflation. Injection of substance P (0.03‐4 pmol) did not alter baseline respiratory pattern. 4. Injections of the EAA antagonists, kynurenic acid (Kyn; 0.6‐240 pmol), 6‐cyano‐7‐nitro‐quinoxaline‐2,3‐dione (CNQX) or 6,7‐dinitroquinoxaline‐2,3‐dione (DNQX) into the BH region of the NTS reversibly impaired the apnoea produced by lung inflation. All three antagonists reduced or abolished the apnoeas resulting from injection of Quis or NMDA, and slowed baseline respiratory frequency. In contrast, injections of the highly selective NMDA receptor antagonist, D‐2‐amino‐5‐phosphonovaleric acids (AP5), in doses sufficient to block the apnoeic response to NMDA, neither altered the reflex apnoea evoked by lung inflation nor the baseline respiratory pattern. 5. Pump cells located within the BH region were excited by pressure injections of the broad spectrum EAA agonist, DL‐homocysteic acid (DLH). Kyn reversibly blocked the excitation of pump cells in response to either lung inflation or DLH injection. 6. These findings suggest that EAAs mediate primary afferent excitation of second‐order neurones in the Breuer‐Hering reflex pathway, primarily through the activation of non‐NMDA EAA receptor subtypes.
Whole‐cell recordings of inwardly rectifying K+ currents activated by 5‐HT1A receptors on dorsal raphe neurones of the adult rat.
Authors: Penington N J , Kelly J S , Fox A P
Published: December 2014

1. An inwardly rectifying K+ current activated by serotonin (5‐HT) was recorded from acutely isolated adult dorsal raphe (DR) neurones using the whole‐cell recording mode of the patch clamp technique. 2. The 5‐HT‐induced K+ current (I5‐HT) was only visible at an [K+]0 > 5 mM and it was observed in 69% of the cells. 3. The reversal potential for I5‐HT was close to the potassium equilibrium potential and was shifted by 51 mV per 10‐fold change in [K+]0 indicating that I5‐HT was carried predominantly by K+. The chord conductance of I5‐HT at ‐90 mV was proportional to the external [K+] raised to a fractional power. 4. A dose‐response relationship revealed that I5‐HT was activated with an ED50 of 30 nM. Ba2+ (0.1 mM) blocked I5‐HT completely. Spiperone reversibly antagonized the response to 5‐HT and 8‐OHDPAT (8‐hydroxy‐2‐(di‐n‐propylamino)tetralin) mimicked the response indicating that the receptor activated was of the 5‐HT1A subtype. 5. The response to 5‐HT was largely prevented by in vitro pretreatment of the cells with pertussis toxin (PTX) indicating the involvement of a PTX‐sensitive G‐protein in the transduction mechanism. 6. cAMP and lipoxygenase metabolites, both implicated in the modulation of similar currents in other preparations, were found not to alter the effectiveness of 5‐HT. 7. Glibenclamide and tolbutamide, blockers of the ATP‐regulated K+ channel, did not reduce the effect of 5‐HT in DR neurones. 8. These results show that in acutely isolated adult DR neurones 5‐HT activates an inwardly rectifying K+ current and this involves a PTX‐sensitive G‐protein in the transduction pathway which may interact with the K+ channel directly.
Power production and working capacity of rabbit tibialis anterior muscles after chronic electrical stimulation at 10 Hz.
Authors: Jarvis J C
Published: December 2014

1. The muscles of the distal anterior compartment of the left hindlimb of rabbits were subjected to continuous indirect electrical stimulation at 10 Hz for periods of up to 12 weeks by means of an implantable stimulator. 2. The maximum shortening velocity (Vmax) and the velocity for maximum power production in single contractions (Vopt) were reduced to 42% and 32% of control values respectively after 12 weeks of stimulation. The rate of change of these parameters was greatest between the second and sixth week of stimulation. These changes, it is suggested, reflect the documented time course of the replacement of fast with slow isoforms of myosin. 3. The reductions in force production and speed of the stimulated muscles combined to produce a marked, progressive decline in the maximum power produced in single contractions. After 8 weeks of stimulation, the maximum power output had fallen to less than 10% of the control value. 4. The fatigue resistance of the stimulated and control muscles was tested over several hours of cyclical shortening contractions designed to elicit an initial power output of 10 W kg‐1 with the muscles set to contract at Vopt. This level of work output represented about 1.6% (control) and 25% (12‐week‐stimulated) of the absolute maximum power output achieved during single contractions. 5. Despite the large reduction in the maximum power output of single contractions, the stimulated muscles showed less than 10% reduction in their power output during the fatigue tests over periods of up to 7 h. The control muscles showed a 70% reduction over the same period. There was no difference in the fatigue resistance under this protocol between muscles stimulated for 2 weeks and those stimulated for longer periods. Transformation of myosin isoforms, which is known to occur later than 2 weeks after the start of stimulation, is not necessary for the induction of this degree of fatigue resistance.
Microheterogeneity of subsarcolemmal sodium gradients. Electron probe microanalysis in guinea‐pig ventricular myocytes.
Authors: Wendt‐Gallitelli M F , Voigt T , Isenberg G
Published: December 2014

1. The effect of stimulation on possible subsarcolemmal sodium accumulation was studied in ventricular myocytes (2 mM [Ca2+]o, 36 degrees C). By trains of eighteen paired voltage‐clamp pulses (180 ms to 0 mV, 20 ms to ‐45 mV, 180 ms to +50 mV, 620 ms to ‐45 mV) unloaded contractions were potentiated to an optimum. 2. Potentiation reversibly enlarged and prolonged the diastolic tail currents due to Na(+)‐Ca2+ exchange. Eighteen pulse pairs were estimated to provide a sodium influx that could increment the total intracellular sodium concentration (sigma Na(i)) by no more than 0.5 mM. 3. Potentiation reversibly increased the current at +50 mV and made it more noisy. Cell‐attached recordings with a second electrode attributed this noise to the activation of K+ (Na) channels. In inside‐out patches, a comparable channel activity was obtained with 40 mM sodium. Hence, the cell‐attached recordings suggest that potentiation can increase intracellular sodium concentration to 40 mM. 4. Electron probe microanalysis (EPMA) measured sigma Na in a volume within 20 nm of the inner side of the sarcolemma. Potentiation reversibly increased sigma Na20nm to 40 +/‐ 7 mM. When stimulation was terminated, sigma Na20nm fell within 8 s to 37 +/‐ 8 mM and within 3 min to 19 +/‐ 6 mM. In unstimulated cells sigma Na20nm was 17 +/‐ 5 mM. 5. In potentiated cells, shock‐frozen at early systole, sigma Na fell with a space constant of 28 nm from the sarcolemma to the centre; at 1 microns distance sigma Na was 12 +/‐ 3 mM. The steep gradient suggests that sodium does not freely diffuse and sigma Na20nm is controlled by transmembrane fluxes rather than by cell dialysis. 6. sigma Na20nm data were distributed with peaks at 5, 30 and 60 mM. Quantitative elemental digital imaging demonstrated patches with 60‐80 mM sigma Na20nm alternating with others of 0‐15 mM sigma Na20nm. This 'sodium microheterogeneity' suggests that Ca2+ efflux at low sigma Na20nm and K+(Na) channel activation at high sigma Na20nm can operate simultaneously.
The physiological regulation of synaptic inhibition by GABAB autoreceptors in rat hippocampus.
Authors: Davies C H , Collingridge G L
Published: December 2014

1. Intracellular recording techniques were used to study the effects of repetitive stimulation on monosynaptically activated inhibitory postsynaptic currents (IPSCs) in rat hippocampal slices. This was achieved by stimulation in stratum radiatum close to a recorded CA1 pyramidal neurone after pharmacological blockade of excitatory synaptic responses, using a combination of the N‐methyl‐D‐aspartate (NMDA) and alpha‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate (AMPA) receptor antagonists D‐2‐amino‐5‐phosphonopentanoate (AP5; 0.04‐0.1 mM) and 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX; 0.02‐0.04 mM), respectively. 2. Fixed‐intensity stimulation at frequencies of less than 0.1 Hz evoked biphasic IPSCs of constant amplitude and waveform. In contrast, when two shocks (paired pulse) or longer trains of ten or more stimuli (i.e. tetani) were delivered at frequencies of between 0.2 and 20 Hz there was marked depression of both phases of every IPSC (by 60‐100%) relative to the first or ‘priming’ IPSC evoked. 3. The gamma‐aminobutyric acid (GABA)B receptor antagonists phaclofen (0.4‐2 mM), 2‐hydroxy‐saclofen (0.02‐0.4 mM) and 3‐aminopropyl(diethoxymethyl)phosphinic acid (CGP 35348; 0.01‐1 mM) reduced or abolished, in a concentration‐dependent and reversible manner, both the late phase of the IPSC (IPSCB) and paired‐pulse depression of the early phase of the IPSC (IPSCA). Expressed in terms of IC50 values, all three antagonists were 5‐10 times more potent at blocking IPSCB than paired‐pulse depression. 4. Paired‐pulse depression, at 5 and 10 Hz, has been shown to be mediated by GABA acting on presynaptic GABAB receptors (i.e. GABAB autoreceptors). We now show that GABAB receptor antagonists reverse paired‐pulse depression over the entire range of frequencies (0.1‐50 Hz) that it occurs. 5. GABAB receptor antagonists reversed substantially the depression of IPSCs during tetani delivered at 5 or 10 Hz. However at 20 Hz, GABAB receptor antagonists appeared to be less effective. At 100 Hz they appeared to be ineffective at reversing the depression of IPSCA; since the antagonists block IPSCB the net effect was to reduce the level of outward current. 6. At frequencies of 20 Hz or more, there was also the appearance of a slow inward current which increased in size in proportion to the frequency and number of shocks in the tetanus. This current (termed here IPSCI) was more pronounced at hyperpolarized membrane potentials and was blocked by picrotoxin (0.1 mM) or bicuculline (0.05 mM). 7. ‘Priming’ is considered to represent a more physiological pattern of activity than a tetanus.(ABSTRACT TRUNCATED AT 400 WORDS)
Physiology, morphology and detailed passive models of guinea‐pig cerebellar Purkinje cells.
Authors: Rapp M , Segev I , Yarom Y
Published: December 2014

1. Purkinje cells (PCs) from guinea‐pig cerebellar slices were physiologically characterized using intracellular techniques. Extracellular caesium ions were used to linearize the membrane properties of PCs near the resting potential. Under these conditions the average input resistance, RN, was 29 M omega, the average system time constant, tau 0, was 82 ms and the average cable length, LN, was 0.59. 2. Three PCs were fully reconstructed following physiological measurements and staining with horseradish peroxidase. Assuming that each spine has an area of 1 micron 2 and that the spine density over the spiny dendrites is ten spines per micrometre length, the total membrane area of each PC is approximately 150,000 microns 2, of which approximately 100,000 microns 2 is in the spines. 3. Detailed passive cable and compartmental models were built for each of the three reconstructed PCs. Computational methods were devised to incorporate globally the huge number of spines into these models. In all three cells the models predict that the specific membrane resistivity, Rm, of the soma is much lower than the dendritic Rm (approximately 500 and approximately 100,000 omega cm2 respectively). The specific membrane capacitance, Cm, is estimated to be 1.5‐2 muF cm‐2 and the specific cytoplasm resistivity, Ri, is 250 omega cm. 4. The average cable length of the dendrites according to the model is 0.13 lambda, suggesting that under caesium conditions PCs are electrically very compact. Brief somatic spikes, however, are expected to attenuate 30‐fold when spreading passively into the dendritic terminals. A simulated 200 Hz train of fast, 90 mV somatic spikes produced a smooth 12 mV steady depolarization at the dendritic terminals. 5. A transient synaptic conductance increase, with a 1 nS peak at 0.5 ms and a driving force of 60 mV, is expected to produce approximately 20 mV peak depolarization at the spine head membrane. This EPSP then attenuates between 200‐ and 900‐fold into the soma. Approximately 800 randomly distributed and synchronously activated spiny inputs are required to fire the soma. 6. The passive model of the PC predicts a poor resolution of the spatio‐temporal pattern of the parallel fibre input. An equally sized, randomly distributed group of approximately 1% of the parallel fibres, activated within a time window of a few milliseconds, would result in approximately the same composite EPSP at the soma.
Neurotensin excitation of rat ventral tegmental neurones.
Authors: Jiang Z G , Pessia M , North R A
Published: December 2014

1. Whole‐cell patch‐clamp recordings were made from ventral tegmental area neurones in rat midbrain slices in vitro. In principal cells, which are presumed to contain dopamine, neurotensin (< or = 1 microM) caused an inward current at ‐60 mV in thirty of forty‐seven neurones and had no effect on the remainder. In secondary neurones, neurotensin caused an inward current in twelve of thirty‐three cells. 2. The inward current evoked by neurotensin reached a maximum amplitude of about 80 pA, and declined over several minutes when the application was discontinued. The current was most commonly accompanied by a decrease in membrane conductance and reversed polarity at a strongly hyperpolarized potential; this reversal potential was less negative in a higher extracellular potassium concentration. Neurotensin also caused an inward current even in potassium‐free internal and external solutions; this current was accompanied by a conductance increase, reversed close to 0 mV and was inhibited by reduction of the extracellular sodium concentration (from 150 to 20 mM). 3. The inward current was associated with a large increase in noise; this persisted in calcium‐free solutions but was inhibited by low sodium concentration. The increase in noise was more prominent at hyperpolarized potentials. The amplitude of the unitary current underlying the increase in noise was estimated from the ratio of the variance to the mean as about 1.5 pA at ‐100 mV. 4. When the recording was made with an electrode containing guanosine 5'‐thio‐triphosphate, the steady inward current evoked by neurotensin did not reverse when the application was discontinued. When the recording electrode contained pertussis toxin, the action of neurotensin was not different although outward currents evoked by dopamine and baclofen declined with time. 5. It is concluded that neurotensin excites ventral tegmental area neurones by activating a pertussis toxin‐insensitive guanosine nucleotide‐binding protein. This leads to a reduction in membrane potassium conductance and an increase in membrane sodium conductance, the relative contribution of which varies from cell to cell.
GABAergic inactivation of the central pattern generators for locomotion in isolated neonatal rat spinal cord.
Authors: Cazalets J R , Sqalli‐Houssaini Y , Clarac F
Published: December 2014

1. Experiments were performed using an isolated brainstem‐spinal cord preparation from newborn rats, in order to study the GABAergic control of the spinal neuronal networks that generate locomotor rhythms in mammals. Locomotor‐like activities were recorded in the ventral roots, and the various neurochemical compounds were added to the superfusion saline. 2. Bath application of GABA suppressed in a dose‐dependent manner the motor activity induced by an excitatory amino acid N‐methyl‐D,L‐aspartate (NMA). Both the GABAA agonist muscimol and the GABAB agonist baclofen mimicked the effects of GABA, since they either slowed down or stopped the rhythmic activity. 3. Experiments were performed in which the lumbar compartment was superfused separately from the brainstem. Chemical activation of the brainstem by NMA alone failed to induce locomotor‐like activity. When GABAA (bicuculline) and GABAB (phaclofen) antagonists were simultaneously bath applied to the lumbar spinal cord, however, locomotor‐like activity was induced. 4. The GABA uptake inhibitors nipecotic acid and guvacine suppressed the rhythmic motor pattern induced by NMA in a dose‐dependent manner. The effects of nipecotic acid were reversed by bicuculline and phaclofen. 5. Bicuculline added during NMA‐induced locomotor‐like activity greatly increased both the frequency and amplitude of motor bursts, while phaclofen modified only the frequency. 6. The motor pattern depended on the balance between activatory and inactivatory influences, since the rhythmic patterns recorded with low doses of NMA associated with high doses of bicuculline were similar to those elicited by higher doses of NMA associated with low doses of bicuculline.(ABSTRACT TRUNCATED AT 250 WORDS)
GABAergic and glutamatergic synaptic input to identified granule cells in salamander olfactory bulb.
Authors: Wellis D P , Kauer J S
Published: December 2014

1. Whole‐cell patch clamp recording techniques were applied to granule cells in an in vitro salamander olfactory bulb preparation to study their morphology, membrane properties and pharmacology of postsynaptic responses to electrical stimulation of either the olfactory nerve (ON) or medial olfactory tract (MOT). Optical recordings of the same preparations stained with the voltage‐sensitive dye RH414 were also made. 2. Anatomical reconstructions of biocytin‐filled granule cells showed that they extend widespread spine‐bearing dendrites and an axon‐like process that branched within the external plexiform layer. 3. ON or MOT stimulation evoked a long‐lasting depolarization, usually generating only a single action potential, in granule cells studied under standard recording conditions. Bath application of bicuculline methiodide (BMI, a GABAA receptor antagonist, 20 or 25 microM) enhanced the spontaneous and electrically evoked excitatory drive to granule cells. 4. The electrically evoked synaptic responses consisted of both excitatory and inhibitory synaptic inputs. Using symmetrical Cl‐ conditions inside and outside the cell to enhance Cl‐ currents, spontaneous and electrically driven BMI‐sensitive inhibitory postsynaptic currents (IPSCs) were revealed, indicating that granule cells receive GABAergic synaptic input. 5. Bath application of GABA (250 microM to 1 mM) shunted and hyperpolarized granule cells as observed directly from whole‐cell recordings and indirectly from cell‐attached patch single channel recordings. 6. Bath application of the glutamate receptor antagonists 6‐cyano‐2,3‐dihydroxy‐7‐nitroquinoxaline (CNQX, 10 microM) and/or DL‐2‐amino‐5‐phosphonopentanoic acid (DL‐AP5, 100 microM) showed that granule cell dendrodendritic EPSPs are shaped by both non‐NMDA and NMDA receptors. 7. The time course and pharmacological sensitivity of both single granule cell responses and ensemble responses recorded optically in the deeper layers of the bulb correlated well. 8. It is concluded that salamander granule cells integrate several types of synaptic input, may have both dendritic and axonal output, and play a major role in generating voltage‐sensitive dye signals in the olfactory bulb.