bluuebunny/crossref_metadata_2025 huggingface dataset:
N‐ethylmaleimide discriminates between two lysine transport systems in human erythrocytes.
Authors: Devés R , Angelo S , Chávez P
Published: December 2014

1. The sulfhydryl reagent N‐ethylmaleimide (NEM) was shown to inactivate the low affinity lysine transporter in human erythrocytes (system y+) without affecting the high affinity transporter (system y+L). 2. Pre‐treatment of the cells with NEM reduced the rate of entry of L‐[14C]lysine (1 microM) by approximately 50% (maximum effect). 3. NEM (0.2 mM) inhibited the NEM‐sensitive component of the flux with mono‐exponential kinetics. The inactivation rate constant (k, +/‐ S.E.M.) was 0.53 +/‐ 0.027 min‐1 (25 degrees C). The substrate did not protect against inactivation. 4. Lysine self‐inhibition experiments revealed two transport systems in untreated cells (half‐saturation constants Km; +/‐ S.E.M.), 12.0 +/‐ 1.7 microM and 109 +/‐ 15.6 microM) and only one high affinity system in NEM‐treated cells (Km 9.5 +/‐ 0.67 microM), indicating that NEM inactivates system y+. 5. The NEM‐insensitive L‐[14C]lysine influx (system y+L) was inhibited with high affinity by unlabelled neutral amino acids. The inhibition constant for L‐leucine in sodium medium (Ki +/‐ S.E.M.) was 10.7 +/‐ 0.72 microM (37 degrees C). The system was also strongly inhibited by L‐methionine, L‐glutamine and with less affinity by L‐phenylalanine and L‐serine. N‐methyl‐L‐leucine, L‐proline and 2‐amino‐2‐norbornane‐carboxylic acid, a bicyclic analogue of leucine, did not exert a significant effect. 6. Lysine transport through system y+L occurred at the same rate in Na+, K+ or Li+ medium and the binding of lysine to the transporter was unaffected by Na+ replacement. 7. The interaction of system y+L with neutral amino acids was dependent on the cation present in the medium. The inhibition constant for leucine and glutamine increased approximately 90‐ and 60‐fold respectively when Na+ was replaced by K+. Li+ was shown to be a very good substitute for Na+.
Functional coupling between the active transport of glucose and the secretion of intestinal neurotensin in rats.
Authors: Dakka T , Cuber J C , Chayvialle J A
Published: December 2014

1. In this study, the mechanisms involved in the release of neurotensin‐like immunoreactivity (NTLI) by glucose were investigated with the isolated, vascularly perfused rat jejunoileum preparation. 2. Luminal infusion of glucose (1‐250 mM) produced a sharp and sustained release of NTLI in the intestinal venous effluent. The first significant response was observed with 5 mM glucose and the release reached a maximum under 250 mM glucose with a plateau secretion at 500% of basal. 3. There was no significant difference in the ability of galactose and 3‐O‐methylglucose to release NTLI when compared to glucose, but alpha‐methylglucose, mannose, 2‐deoxyglucose and fructose did not stimulate NTLI release. 4. Luminal infusion of 5 mM phloridzin reduced the glucose‐induced release of NTLI by 90%. Intra‐arterial infusion of glucose (25 mM) or of phloretin (20 microM) had no significant effect on the glucose‐evoked NTLI secretion. 5. Intra‐arterial infusion of ouabain (1 mM) produced a dramatic increase (at about 1500% of basal) in portal NTLI although it drastically reduced intestinal absorption of glucose. 6. Intra‐arterial infusion of tetrodotoxin (1 microM), atropine (10 microM), verapamil (50 microM) or nifedipine (50 microM) did not modify the glucose‐induced NTLI secretion. 7. Intra‐arterial infusion of forskolin (2‐20 microM) evoked a prompt and well‐sustained secretion of NTLI which was increased to a mean value of 800% of basal with the highest dose tested. 3‐Isobutyl‐1‐methylxanthine (IBMX, 10‐100 microM) also stimulated the secretion of NTLI (maximal increase at 725% of basal at 100 microM). In contrast, intra‐arterial infusion of 4‐beta‐phorbol 12‐myristate, 13‐acetate (PMA, 0.05‐0.5 microM) had no effect on NTLI release. 8. IBMX (10‐100 microM) synergistically enhanced NTLI responses induced by 250 mM glucose; the integrated response of NTLI release was 3‐ to 5‐fold higher than the sum of individual responses produced by the same stimulants given separately. 9. It is concluded that the carbohydrate‐induced NTLI release is related to the active, sodium‐dependent hexose transport, but not to the carbohydrate catabolic pathway. Furthermore, the intramural nerves and L‐type calcium channels are not involved in the glucose‐induced NTLI secretion. Finally, the secretory activity of the intestinal N cell seems to be mainly stimulated through a cAMP‐dependent pathway.
Expression of subunit‐omitted mouse nicotinic acetylcholine receptors in Xenopus laevis oocytes.
Authors: Liu Y , Brehm P
Published: December 2014

1. Nicotinic acetylcholine (ACh) receptors in developing vertebrate skeletal muscle exhibit functional heterogeneity in both conductance and kinetics. To assess the contributions of receptors differing in subunit composition to the heterogeneity, various combinations of mouse alpha beta delta gamma epsilon subunit RNAs were tested for the ability to express functional receptors in Xenopus oocytes. 2. Two combinations of dual‐subunit RNAs (alpha delta and alpha gamma) resulted in detectable ACh‐activated currents and six different combinations of three or more subunit RNAs produced significant numbers of functional channels. The order of combinations yielding the greatest amount of current was alpha beta gamma > alpha beta delta = alpha delta epsilon > alpha delta gamma > alpha delta > alpha gamma. 3. The extent to which a channel type with three different subunits was expressed was highly dependent upon the ratios of RNAs coding for the different subunits. For alpha beta delta receptors the efficiency of expression was alpha: beta: delta (1/5:1/5:1) >> (1:1:1) >> (1/5:1:1/5) > (1:1/5:1/5). 4. The level of expression of three‐subunit combinations was also critically dependent upon the order of RNAs injected. When alpha delta or alpha gamma RNA combinations were co‐injected 2 days prior to the injection of beta RNA, the expression was 2‐5 times greater than when alpha beta injection was followed by injection of delta or gamma RNA. 5. Single‐channel measurements revealed that alpha beta delta channels were not expressed in the presence of alpha beta delta epsilon RNAs, even under conditions when the amount of delta RNA injected was 5‐fold higher than the amount of epsilon RNA. 6. These data indicate that the functional expression of subunit‐omitted receptors depends critically upon the relative amounts of the five different subunit RNAs. Receptors composed of three different subunits express in the presence of the subunit RNAs characteristic of embryonic muscle (alpha beta delta gamma), but are not observed with the combination of RNAs characteristic of adult muscle (alpha beta delta epsilon).
Rundown of N‐methyl‐D‐aspartate channels during whole‐cell recording in rat hippocampal neurons: role of Ca2+ and ATP.
Authors: Rosenmund C , Westbrook G L
Published: December 2014

1. N‐methyl‐D‐aspartate (NMDA) channel activity was studied on cultured rat hippocampal neurons in whole‐cell voltage‐clamp mode. NMDA responses were evoked by rapid application of NMDA and the cytosol was modified using pipette dialysis and intracellular perfusion. 2. In the presence of 2 mM [Ca2+]o with 2.4 mM BAPTA (1,2‐bis(O‐aminophenoxy)ethane‐N,N,N',N'‐tetraacetic acid) and 0.4 mM Ca2+ in the whole‐cell pipette, the response evoked by regular applications of 10 microM NMDA gradually decreased during prolonged whole‐cell recording. After 25 min the peak current was reduced to 56 +/‐ 1.6% of control. Channel ‘rundown’ could be prevented by inclusion of an ATP regenerating solution in the pipette. 3. Rundown did not occur in Ca(2+)‐free medium even in the absence of added ATP regenerating solution. Rundown was also prevented by increasing [BAPTA]i to 10 mM whereas raising [Ca2+]i by inhibiting the Na(+)‐Ca2+ exchanger or by perfusing the patch pipette with high [Ca2+]i (15‐1000 microM) reversibly inhibited the NMDA current. By contrast, the rundown of kainate responses was Ca(2+)‐independent. 4. The rate and reversibility of rundown was use‐dependent. Rundown did not occur with infrequent NMDA applications (0.2/min). Following channel rundown in Ca(2+)‐containing medium, a 5 min pause in agonist applications or adding ATP regenerating solution by intracellular perfusion resulted in complete recovery. However, rundown did not recover following large currents evoked by 300 microM NMDA or when 10 mM EGTA was used as the intracellular buffer. Protease inhibitors did not prevent irreversible rundown. 5. ATP‐gamma‐S (4 mM) was less effective than the ATP regenerating solution in preventing rundown. Likewise, intracellular dialysis with alkaline phosphatase, phosphatase 1 or calcineurin did not induce rundown and addition of phosphatase inhibitors also did not block rundown. Thus receptor dephosphorylation did not appear to be primarily responsible for channel rundown. 6. The mean open time and unitary conductance of the NMDA channel were unaffected by rundown as estimated by fluctuation analysis. The conductance was 42.8 +/‐ 2.9 nS before and 43.7 +/‐ 2.8 nS after rundown. The mean open times were 17.3 and 4.0 ms before and 15.9 and 4.0 ms after rundown. However the open probability was reduced following rundown as determined by the onset of MK‐801 block of steady‐state NMDA currents. 7. Our results suggest that an increase in intracellular calcium leads to channel rundown during whole‐cell recording by reducing the open probability of the NMDA channel.(ABSTRACT TRUNCATED AT 400 WORDS)
Differential effect of injections of kainic acid into the prepositus and the vestibular nuclei of the cat.
Authors: Godaux E , Mettens P , Cheron G
Published: December 2014

1. In order adequately to control eye movements, oculomotoneurones have to be supplied with both an eye‐velocity signal and an eye‐position signal. However, all the command signals of the oculomotor system are velocity signals. Nowadays, there is general agreement about the existence of a brainstem network that would convert velocity command‐signals into an eye‐position signal. This circuit, because of its function, is called the oculomotor neural integrator. The most obvious symptom of its eventual failure is a gaze‐holding deficit: in this case, saccades are followed by a centripetal post‐saccadic drift. Although the oculomotor neural integrator is central in oculomotor theory, its precise location is still a matter for debate. 2. Previously, microinjections of kainic acid (KA) into the region of the nucleus prepositus hypoglossi (NPH) and of the medial vestibular nucleus (MVN) were found to induce a horizontal gaze‐holding failure both in the cat and in the monkey. However, the relatively large volumes (1‐3 microliters) and concentrations (2‐4 micrograms microliters‐1) used in these injections made it difficult to know if the observed deficit was due to a disturbance of the NPH or of the nearby MVN. These considerations led us to inject very small amounts of kainic acid (50 nl, 0.1 microgram microliter‐1) either into the rostral part of the MVN or into different sites along the NPH of the cat. 3. The search coil technique was used to record (1) spontaneous eye movements (2) the vestibulo‐ocular reflex (VOR) induced by a constant‐velocity rotation (50 deg s‐1 for 40 s) and the optokinetic nystagmus (OKN) elicited by rotating an optokinetic drum at 30 deg s‐1 for 40 s. 4. In each injection experiment, the location of the abducens nucleus of the alert cat was mapped out by recording the antidromic field potentials evoked by the stimulation of the abducens nerve. Two micropipettes were then glued together in such a way that when the tip of the recording micropipette was in the centre of the abducens nucleus the tip of the injection micropipette was in a target area. The twin pipettes were then lowered in the brainstem until the recording micropipette reached the centre of the abducens nucleus. Kainic acid was then injected into the brainstem of the alert cat through the injection micropipette by an air pressure system. 5. Carried out according to such a protocol, KA injections into the NPH or the rostral part of the MVN consistently led to specific eye‐movement changes.(ABSTRACT TRUNCATED AT 400 WORDS)
Implication of frequenin in the facilitation of transmitter release in Drosophila.
Authors: Rivosecchi R , Pongs O , Theil T , Mallart A
Published: December 2014

1. We have investigated the possible role of frequenin in the modulation of synaptic facilitation at the larval Drosophila neuromuscular junctions. Excitatory junctional currents (EJCs) and presynaptic nerve terminal currents were recorded by external electrodes in normal larvae and in transgenic larvae carrying an extra insertion of the frequenin cDNA. 2. Motor nerve stimulation by twin pulses or trains of stimuli provoked EJC facilitation which was about three times higher in transgenic larvae compared to controls. Unconditioned EJCs revealed, however, similar quantal content and Ca2+ sensitivity in both Drosophila strains. 3. Differences between normal and transgenic Drosophila in the quantal content of the facilitated EJC do not depend on differences in the duration of the repolarization phase of the presynaptic action potential. 4. Perfusion of tetrodotoxin or of low‐Na+ solutions abolished the enhancement of the EJC facilitation observed in the transformants. These treatments only slightly affected the facilitation of normal junctions. 5. These results suggest that (i) internal Na+ accumulation can enhance facilitation of transmitter release in Drosophila neuromuscular junctions overexpressing frequenin, and (ii) this effect possibly depends on a modulation of the activity of the Na(+)‐Ca2+ exchanger by frequenin.
Synaptic transmission between single slowly adapting type I fibres and their cuneate target neurones in cat.
Authors: Vickery R M , Gynther B D , Rowe M J
Published: December 2014

1. The synaptic linkage between single, identified slowly adapting type I (SAI) fibres and their central target neurones of the cuneate nucleus was examined in pentobarbitone‐anaesthetized cats. Simultaneous extracellular recordings were made from individual cuneate neurones and from fine, intact fascicles of the lateral branch of the superficial radial nerve in which it was possible to identify and monitor the activity of each group II fibre. Individual SAI fibres were activated by static displacement and by vibration delivered with a fine probe (0.25‐2 mm diameter) to their associated touch domes in the hairy skin of the forelimb. 2. Transmission properties across the synapse were analysed for nine SAI‐cuneate pairs in which the single SAI fibre of each pair provided a suprathreshold input to the cuneate neurone. Neither spatial nor temporal summation was required for effective impulse transmission, and often more than 80% of SAI impulses led to a response in the cuneate neurone. Responses of the cuneate neurones to single SAI impulses occurred at a short, fixed latency (S.D. often < 0.1 ms), and frequently consisted of a burst of two or three impulses, at low SAI input rates in particular. 3. The tight phase‐locking in the responses to vibration of single SAI fibres was preserved in the cuneate responses for frequencies up to approximately 400 Hz. However, as the impulse rates of the cuneate neurones were less than 150 impulses s‐1, their impulse patterns could not directly signal the vibration periodicity at frequencies > 100‐150 Hz despite 1:1 responses in their single SAI input fibres up to approximately 500 Hz. 4. The reliable transmission of touch dome‐associated SAI input across the cuneate nucleus indicates that transmission failure at this first relay is unlikely to be responsible for the reported failure of touch dome‐SAI inputs to contribute to tactile perception. 5. The transmission characteristics for the SAI fibres were very similar to those demonstrated previously for fibres associated with Pacinian corpuscles, which argues against any marked differential specialization in transmission characteristics for dorsal column nuclei neurones that receive input from different tactile fibre classes.
Calcium‐dependent chloride current induced by axotomy in rat sympathetic neurons.
Authors: Sánchez‐Vives M V , Gallego R
Published: December 2014

1. Seven to ten days after sectioning their axons, rat sympathetic neurons were studied using intracellular recording techniques in an in vitro preparation of the superior cervical ganglion. 2. In 75% of axotomized cells, an after‐depolarization (ADP) was observed following spike firing or depolarization with intracellular current pulses. Discontinuous single‐electrode voltage‐clamp techniques were employed to study the ADP. When the membrane potential was clamped at the resting level just after an action potential, a slow inward current was recorded in cells that showed an ADP. 3. In the presence of TTX and TEA, inward peaks and outward currents were recorded during depolarizing voltage jumps, followed by slowly decaying inward tail currents accompanied by large increases in membrane conductance. The inward peak and tail currents activated between ‐10 and ‐20 mV and reached maximum amplitudes around 0 mV. With depolarizing jumps to between +40 and +50 mV, net outward currents were recorded during the depolarizing jumps but inward tail currents were still activated. 4. In the presence of the Ca2+ channel blocker cadmium, or when Ca2+ was substituted by Mg2+, the ADP disappeared. In voltage‐clamped cells, cadmium blocked the inward tail currents. The reversal potential for the inward tail current was approximately ‐15 mV. Substitution of the extracellular NaCl by sucrose or sodium isethionate increased the amplitude of the inward tail current, and displaced its equilibrium potential to more positive values. Changes in extracellular [K+] did not appreciably affect the inward tail current amplitude or equilibrium potential. Niflumic acid, a blocker of chloride channels activated by Ca2+, almost completely blocked the tail current. 5. No ADPs were observed in non‐axotomized neurons, and when depolarizing pulses were applied while in voltage clamp no inward tail currents were evoked in these normal cells. 6. It is concluded that axotomy of sympathetic ganglion cells produces the appearance of a Ca(2+)‐dependent chloride current responsible for the ADP observed following spike firing.
Two distinct glutamatergic synaptic inputs to striatal medium spiny neurones of neonatal rats and paired‐pulse depression.
Authors: Mori A , Takahashi T , Miyashita Y , Kasai H
Published: December 2014

Excitatory postsynaptic currents (EPSCs) were recorded from the medium spiny neurones of neonatal rat striatal slices using the whole‐cell patch clamp method. EPSCs were selectively elicited in the presence of picrotoxin with a glass stimulating pipette placed in the striatum. We found two distinct unitary EPSCs that were evoked by stimulation of single presynaptic fibres. The major type of EPSC, termed ‘S‐type’, failed frequently and had a small mean amplitude (2.05 pA). They probably represented cortical afferents. The other type of unitary EPSC, the ‘H‐type’, seldom failed and was 13 times larger than the S‐type. Spontaneous EPSCs with amplitudes similar to those of H‐type EPSCs could be induced. H‐type EPSCs were mediated by both non‐NMDA and NMDA receptors. The two types of EPSCs could be evoked in the same neurons. The intensity of stimulation for H‐type EPSCs was higher than that for S‐type EPSCs. H‐type EPSCs could be polysynaptically activated, suggesting the presence of glutamatergic interneurones in the striatum that generated H‐type EPSCs. H‐type EPSCs displayed particularly long‐lasting paired‐pulse depression, while that displayed by the S‐type EPSCs was short. The paired‐pulse depression of both EPSCs was Ca2+ dependent and involved presynaptic mechanisms. We have demonstrated that the medium spiny neurones of neonatal rats receive two different glutamatergic input systems having different amplitudes, origins and paired‐pulse depression, reminiscent of cerebellar Purkinje cells. This suggests that the two types of EPSCs also play distinctive roles in striatal neuronal circuitry.
Single channel properties of cloned NMDA receptors in a human cell line: comparison with results from Xenopus oocytes.
Authors: Stern P , Cik M , Colquhoun D , Stephenson F A
Published: December 2014

1. Human embryonic kidney (HEK) 293 cells were transiently transfected with cDNAs encoding the NR1a‐NMDA epsilon 1[NR2A] subunit combination of the NMDA receptor. Single channel behaviour was recorded from outside‐out membrane patches, with the aim of comparing the results with those, recorded under the same conditions, from Xenopus oocytes injected with messenger RNA coding for the NR1a‐NR2A combination. 2. Single channels in HEK 293 cells showed a main conductance level of 51.4 +/‐ 2.4 pS, compared with 50.1 +/‐ 1.4 pS for channels in oocytes. A subconductance level of 38.1 +/‐ 2.1 pS was found in HEK 293 cells, compared with 38.3 +/‐ 1.3 pS in oocytes. The frequencies of transitions between the shut and the two conductance levels were also very similar. 3. Distributions of shut times could be fitted with five exponential components. In HEK 293 cells the first three of these components had time constants of 39 +/‐ 4 microseconds, 0.54 +/‐ 0.04 ms and 9.94 +/‐ 1.3 ms; in oocytes the values were 69 +/‐ 35 microseconds, 0.54 +/‐ 0.15 ms and 6.53 +/‐ 4.6 ms, respectively. The relative areas of the components were also similar in the two systems. 4. The distribution of all apparent open times for the sublevels was fitted with two exponential components giving time constants of 0.18 +/‐ 0.02 ms and 1.31 +/‐ 0.17 ms (for HEK cells) or of 0.31 +/‐ 0.36 ms and 1.31 +/‐ 1.1 ms (for oocytes).(ABSTRACT TRUNCATED AT 250 WORDS)
Nicotinic antagonist‐produced frequency‐dependent changes in acetylcholine release from rat motor nerve terminals.
Authors: Tian L , Prior C , Dempster J , Marshall I G
Published: December 2014

1. The frequency (0.5‐150 Hz) and calcium dependence (0.5‐2.0 mM) of the effects of the nicotinic antagonist tubocurarine (0.2 microM) on acetylcholine (ACh) liberation from motor nerve terminals has been examined using binomial analysis of quantal transmitter release. 2. At an extracellular calcium ion concentration ([Ca2+]o) of 2.0 mM, tubocurarine produced a decrease in the endplate current (EPC) quantal content of approximately 30% at high frequencies of motor nerve stimulation (50‐150 Hz). In contrast, at low frequencies of stimulation (0.5‐1.0 Hz), tubocurarine enhanced the EPC quantal content by approximately 20%. 3. The enhancement of EPC quantal content produced by tubocurarine at low frequencies of motor nerve stimulation was [Ca2+]o dependent, being abolished when [Ca2+]o was lowered from 2.0 to 0.5 mM. In contrast, the decrease in quantal content produced by tubocurarine at high frequencies of motor nerve stimulation was independent of [Ca2+]o, being approximately 30% at all calcium ion concentrations studied. 4. In direct contrast to tubocurarine, the nicotinic antagonist vecuronium (1.0 microM) produced no increase in EPC quantal content at low frequencies of nerve stimulation. However, at high frequencies of nerve stimulation it decreased EPC quantal content to a similar extent to 0.2 microM tubocurarine. The frequency‐dependent decrease in EPC quantal content produced by 1.0 microM vecuronium in 2.0 mM [Ca2+]o was very similar to that seen with 0.2 microM tubocurarine in 0.5 mM [Ca2+]o. 5. Binomial analysis revealed that all the changes in EPC quantal content associated with both nicotinic antagonists were due to changes in the size of the pool of quanta in the nerve terminal available for immediate release with no effect on the probability of release of an individual quantum. 6. The results are interpreted in terms of two separately identifiable prejunctional actions of the nicotinic antagonists, both involving an action at nicotinic ACh receptors situated on the motor nerve terminal. Thus, at high frequencies of motor nerve stimulation tubocurarine and vecuronium produce a [Ca2+]o‐independent decrease in ACh release, probably through an inhibitory action on a positive‐feedback prejunctional nicotinic autoreceptor closely related to the muscle‐type nicotinic ACh autoreceptor. However, at low frequencies of motor nerve stimulation we suggest that tubocurarine, but not vecuronium, produces a [Ca2+]o‐dependent increase in ACh release through an action at a negative‐feedback prejunctional neuronal‐type nicotinic ACh autoreceptor.
Position‐dependent expression of potassium currents by chick cochlear hair cells.
Authors: Murrow B W
Published: December 2014

1. Potassium currents in chick cochlear hair cells were studied using whole‐cell voltage clamp techniques. Cells were isolated from 200 microns‐long segments of the apical half of the cochlea. In each segment, expression of potassium currents by cells positioned across the width (‘inner‐outer’ hair cell axis) of the cochlea was examined. 2. A rapidly inactivating potassium current (IA) was found in some hair cells. At a membrane potential of ‐24 mV, IA activated to peak values within 7 +/‐ 1 ms and inactivated within 73 +/‐ 16 ms. The activation ‘threshold’ was around ‐50 mV and hyperpolarization more negative than ‐56 +/‐ 5 mV was required before significant removal of inactivation occurred (V 1/2 (half‐inactivation potential) = ‐74 +/‐ 5 mV). The resting potential of cells with IA was ‐46 mV +/‐ 11 mV. This current was blocked by 4‐aminopyridine with a Kd of 0.45 mM. 3. Cells that were isolated from the most apical tip of the cochlea expressed no IA. In areas more basal than 200 microns from the apex, the magnitude of IA correlated with cell morphology. In each area, the tallest hair cells (cells with the smallest ratio of apical surface diameter to length) had none of this current. Of the cells with IA, the shorter cells (larger ratio of apical surface diameter to length) had more of this current. 4. The magnitude of IA in a cell was dependent upon cross‐cochlear position, and the relationship between IA and cell morphology was most probably a reflection of a differential distribution of cell shape across the cochlea. The tallest hair cells, occupying roughly the first 40% of the distance from the neural side of the basilar papilla, had no IA. Of the remaining cells, those nearer to the abneural edge expressed more IA, such that iso‐magnitude lines ran approximately parallel to the long axis of the cochlea. 5. A delayed rectifier current (IK) and an inward rectifier current (IIR) were also differentially distributed among hair cells across the cochlea; however, their distribution differed from that of IA. IK and IIR were preferentially expressed by the taller hair cells, which were positioned nearer to the neural side of the cochlea. Ca(2+)‐activated potassium current (IK(Ca)) did not vary systematically between cells of different shape or cross‐cochlear position, and IK(Ca) could often be found in cells with IA.
Activity of bulbar respiratory neurons during fictive coughing and swallowing in the decerebrate cat.
Authors: Oku Y , Tanaka I , Ezure K
Published: December 2014

1. The behaviour of medullary respiratory neurons was studied during fictive coughing and swallowing evoked by electrical stimulation of the superior laryngeal nerve (SLN) in decerebrate, paralysed and artificially ventilated cats. Fictive coughing, swallowing and respiration were monitored by recording activities of the phrenic, hypoglossal and abdominal nerves. 2. Extracellular recordings were made from respiratory neurons in the ventral respiratory group (VRG) and in the Bötzinger complex (BOT). The neuronal types analysed included decrementing inspiratory neurons (I‐DEC), augmenting expiratory neurons (E‐AUG) and decrementing expiratory neurons (E‐DEC) from the BOT area, and augmenting inspiratory neurons (I‐AUG) and augmenting expiratory neurons (E‐AUG) from the VRG area. 3. During fictive coughing, all the inspiratory and expiratory neurons were active during the inspiratory and expiratory phases of coughing, respectively. The firing of both I‐DEC and I‐AUG neurons was increased and prolonged in association with the augmented inspiratory activity of the phrenic nerve. The activity of E‐AUG neurons of the VRG did not parallel the abdominal nerve activity, suggesting the existence of additional neurons which participate in the generation of abdominal nerve activity during fictive coughing. 4. During fictive swallowing, half of I‐DEC neurons fired transiently at the onset of hypoglossal bursts associated with swallowing; the firing was suppressed during the rest of the hypoglossal bursts. Other I‐DEC neurons were silent during hypoglossal bursts. Some I‐AUG neurons fired during the initial half of hypoglossal bursts, and others were silent. The brief phrenic activity accompanying the swallowing might have originated from this activity in I‐AUG neurons. The discharges of all E‐AUG neurons (BOT and VRG) and the majority of E‐DEC BOT neurons were suppressed during swallowing. 5. We conclude that these five types of respiratory neurons of the BOT and VRG are involved in the generation of the spatiotemporally organized activity of coughing and swallowing, and that at least a part of the neuronal network for respiration is shared by networks for these non‐respiratory activities.
Neuronal regulation of cochlear blood flow in the guinea‐pig.
Authors: Laurikainen E A , Costa O , Miller J M , Nuttall A L , Ren T Y , Masta R , Quirk W S , Robinson P J
Published: December 2014

1. Previous studies have shown that electrical stimulation (ES) of the guinea‐pig cochlea causes a neurally mediated increase in cochlear blood flow (CBF). It is known that the centrifugal neuronal input to the cochlea comes through the perivascular sympathetic plexus from the cervical sympathetic chain and along the vestibular nerve (VN) from the periolivary area of the brainstem. Both of these neuronal systems are distributed topographically in the cochlea. 2. In order to study the neural origins of ES‐evoked CBF increase, laser Doppler flowmetry was used to test the following hypotheses. (a) The response is regional, that is, limited to the area of the cochlea stimulated. To test this we performed differential ES of the cochlear turns. CBF was measured from either the third or the first turn. (b) The response is mediated via autonomic receptors within the cochlea. To study this, we applied atropine, succinylcholine and idazoxan locally to the cochlea. (c) The response is influenced by neuronal input via the sympathetic cervical chain (SC) and components of the VN. We stimulated and sectioned the SC, and sectioned the VN, to test this hypothesis. 3. We observed that the CBF response was topographically restricted to the stimulated region. Locally applied muscarinic or nicotinic antagonists (atropine and succinylcholine respectively) did not affect the response. However, local idazoxan (an alpha 2‐blocker) eliminated the response. Locally applied adrenaline and SC stimulation modified the dynamic range of the response. SC sectioning enhanced the responsiveness of the cochlear vasculature to ES. The VN section caused a temporary decrease in CBF and elimination of the ES‐evoked CBF response. 4. We conclude that the release of dilating agents is topographical with respect to ES current flow, the ES‐evoked CBF increase is peripherally mediated via alpha 2‐receptors, and the response is influenced by input via the SC. The elimination of the response by VN sectioning proximal to the brainstem indicated that fibres of the VN mediate the CBF increase during direct cochlear ES. The data suggest that these fibres may be the efferent limb of a neural loop involved with the regulation of CBF. Such a system could provide a mechanism for the rapid increase in CBF with organ stress.
Medullary loci critical for expression of gasping in adult rats.
Authors: Fung M L , Wang W , St John W M
Published: December 2014

1. Our purpose was to define whether a region of medulla could be identified that is critical for the expression of gasping. 2. Decerebrate, vagotomized, paralysed and ventilated adult rats were used. The pattern of phrenic activity was reversibly altered from eupnoea to gasping by exposure to hypoxia or anoxia. 3. Gasping was irreversibly eliminated following unilateral electrolytic lesions of the lateral tegmental field of the medulla. The eupnoeic rhythm continued after these lesions. 4. Injections of kainic acid into the lateral tegmental field also eliminated gasping. Phrenic activity in eupnoea was not altered. 5. Lesions outside the lateral tegmental field caused marked changes in the eupnoeic rhythm, including expiratory apnoea. Upon exposure to hypoxia or anoxia, gasping was still induced. 6. This region for the neurogenesis of gasping in rats is identical to the region that serves a comparable function in cats. Moreover, it overlaps with the ‘pre‐Bötzinger’ complex which has been described for the in vitro brainstem preparation of the neonatal rat. Our results raise doubts that this complex plays a role in the neurogenesis of eupnoea.
Reversal of rectification and alteration of selectivity and pharmacology in a mammalian Kv1.1 potassium channel by deletion of domains S1 to S4.
Authors: Tytgat J , Vereecke J , Carmeliet E
Published: December 2014

1. A possible relation between the family of inwardly rectifying K+ channels and the Shaker superfamily of K+ channels was investigated using a deletion mutant (DelS1‐S4) of a delayed rectifier Kv1.1 (RCK1) K+ channel. 2. The mutant DelS1‐S4 was made by eliminating the sequence coding for transmembrane domains S1 to S4 of the Kv1.1 K+ channel, and re‐ligating the sequence coding for the cytoplasmic amino terminus to transmembrane domain S5. Microelectrode voltage‐clamp and patch‐clamp experiments were performed on Xenopus laevis oocytes after injection of in vitro transcribed mRNA coding for mutant and wild‐type channels. 3. The lack of transmembrane domains S1 to S4 converts a depolarization‐activated wild‐type Kv1.1 K+ channel with outward rectification into a hyperpolarization‐activated channel with inward rectification. Although the pore region of the deletion mutant is identical to the wild‐type channel, the mutant channel is a non‐selective cation channel and is characterized by an altered pharmacology profile.
Excitatory synaptic connections onto rat hippocampal inhibitory cells may involve a single transmitter release site.
Authors: Arancio O , Korn H , Gulyas A , Freund T , Miles R
Published: December 2014

1. Whole‐cell tight‐seal records of excitatory postsynaptic currents (EPSCs) were made from inhibitory cells in the CA3 region of thin hippocampal slices. We tested the hypothesis that excitatory synaptic connections made on inhibitory cells involve few transmitter release sites. 2. EPSCs impinging on inhibitory cells had a time to peak of 0.4‐3.8 ms and an amplitude of 8‐90 pA at a holding potential of ‐60 mV. They were suppressed by the excitatory amino acid antagonists 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) and DL‐2‐amino‐5‐phosphonovaleric acid (APV). 3. Addition of tetrodotoxin (TTX) and Co2+ to the external solution reduced the frequency of EPSCs from 0.90 to 0.25 s‐1 (n = 24 cells). In the majority of cells EPSC amplitude distributions were not significantly changed. 4. Increasing Ca2+ and reducing Mg2+ in the external solution, in order to enhance the probability of transmitter release, did not change EPSC amplitude distributions. In contrast, amplitude histograms for IPSCs recorded from pyramidal cells were shifted to higher mean values in this solution. 5. EPSCs were elicited in inhibitory cells by electrical stimulation via a glass pipette placed near to pyramidal cells in stratum pyramidale. EPSCs elicited by weak stimuli had similar amplitude distributions to excitatory synaptic events recorded in the presence of TTX and Co2+. 6. These findings suggest excitatory synaptic connections made with CA3 inhibitory cells involve few or possibly just one transmitter release site.
Effects of creatine phosphate and P(i) on Ca2+ movements and tension development in rat skinned skeletal muscle fibres.
Authors: Fryer M W , Owen V J , Lamb G D , Stephenson D G
Published: December 2014

1. Mechanically skinned fast‐twitch (FT) and slow‐twitch (ST) muscle fibres of the rat were used to investigate the effects of fatigue‐like changes in creatine phosphate (CP) and inorganic phosphate (P(i)) concentration on Ca(2+)‐activation properties of the myofilaments as well as Ca2+ movements into and out of the sarcoplasmic reticulum (SR). 2. Decreasing CP from 50 mM to zero in FT fibres increased maximum Ca(2+)‐activated tension (Tmax) by 16 +/‐ 2% and shifted the mid‐point of the tension‐pCa relation (pCa50) to the left by 0.28 +/‐ 0.03 pCa units. In ST fibres, a decrease of CP from 25 mM to zero increased Tmax by 9 +/‐ 1% and increased the pCa50 by 0.16 +/‐ 0.01 pCa units. The effect of CP on Tmax was suppressed in both fibre types by prior treatment with 0.3 mM FDNB (1‐fluoro‐2,4‐dinitrobenzene), suggesting that these effects may occur via changes in creatine kinase activity. 3. Increases of P(i) in the range 0‐50 mM reduced the pCa50 and Tmax in both fibre types. These effects were more pronounced in ST fibres than in FT fibres in absolute terms. However, normalization of the results to resting P(i) levels appropriate to both fibre types (1 mM for FT and 5 mM for ST fibres) revealed similar decreases in Tmax (approximately 39% at 25 mM P(i) and approximately 48% at 50 mM P(i)) and pCa50 (0.25 pCa units at 25‐50 mM P(i)). The depressant action of P(i) on both parameters was considerably reduced when the rise in P(i) was accompanied by an equivalent reduction in [CP]. 4. Tension development in the presence of complex, fatigue‐like milieu changes (40 mM P(i) for FT; 20 mM P(i) for ST) was decreased by 35‐40% at a constant myoplasmic [Ca2+] of 6 microM in both fibre types. 5. SR Ca2+ loading at a myoplasmic [Ca2+] of 100 nM was found to increase abruptly when the [P(i)] during loading was increased to near 9 mM. At a myoplasmic [Ca2+] of 300 nM, the threshold P(i) for this effect dropped to approximately 3 mM. 6. Tension responses evoked by caffeine in the absence of P(i) were smaller and slower to peak if fibres were exposed to P(i) in a restricted myoplasmic Ca2+ pool after SR Ca2+ loading. This indicated that myoplasmic P(i) can decrease and prolong the rate of Ca2+ release from the SR.(ABSTRACT TRUNCATED AT 400 WORDS)
Stimulation of guinea‐pig tracheal afferent fibres by non‐isosmotic and low‐chloride stimuli and the effect of frusemide.
Authors: Fox A J , Barnes P J , Dray A
Published: December 2014

1. Inhalation of low‐chloride or non‐isosmotic solutions evokes cough or reflex bronchoconstriction in humans that is inhibited by frusemide (furosemide), whilst capsaicin‐evoked cough is unaffected. Here we have examined the responses of single vagal afferent fibres innervating the isolated guinea‐pig trachea to these stimuli, and tested the effect of frusemide on fibre responses. 2. Both distilled water and hypertonic saline applied for 30 s onto identified receptive fields produced marked excitation of all A delta and C fibres tested. Isotonic glucose, a low‐chloride solution, was a less potent stimulant and caused excitation in 37% of A delta fibres and 69% of C fibres. There was no difference in the distribution of low‐chloride sensitive and insensitive receptive fields. 3. In the presence of frusemide, responses of A delta fibres to isotonic glucose were significantly inhibited to 34.2 +/‐ 6.2% of the pre‐drug control level. However, frusemide was without effect either on responses of A delta fibres to distilled water or hypertonic saline, or on responses of C fibres to capsaicin. 4. These data support a role for tracheo‐bronchial A delta and C fibres in airway reflexes evoked by hypotonic, hypertonic and low‐chloride stimuli. The protective effect of frusemide against airway responses to low‐chloride but not to non‐isosmotic solutions may reflect an action on sensory nerve endings.
Suppression of GABAA receptor responses by NMDA application in hippocampal neurones acutely isolated from the adult guinea‐pig.
Authors: Chen Q X , Wong R K
Published: December 2014

1. In acutely isolated hippocampal cells, NMDA and glutamate application suppressed GABAA receptor‐mediated responses. We studied the cellular events underlying the interaction between the two classes of receptors by using a whole‐cell voltage‐clamp approach. 2. Following an NMDA application, an outward current mediated by GABAA receptor activation (GABA response) was suppressed for up to 12 s. The suppression of the GABA response was reduced when Ca2+ in the extracellular solution was replaced by Ba2+ or when intracellular BAPTA (1,2‐bis(O‐aminophenoxy)ethane‐N,N,N',N'‐tetraacetic acid) was increased from 1 to 10 mM. 3. Replacing ATP in the intracellular solution by adenosine‐5'‐O‐3‐thiotriphosphate reduced the suppressive effect of NMDA application on the GABA response. Okadaic acid, a phosphatase inhibitor, also prevented the NMDA‐induced suppression of the GABA response. In addition, when the intracellular perfusing solution contained the calcineurin autoinhibitory fragment (50 microM), suppression of the GABA response by the NMDA current was also reduced. 4. Intracellular perfusion of an activated form of the Ca(2+)‐dependent phosphatase, calcineurin, suppressed GABA responses. 5. The results show that NMDA responses elicited in hippocampal neurones transiently suppressed GABA responses. The data suggest that the functional linkage of the NMDA response with the GABA response was established via a Ca(2+)‐dependent dephosphorylation process.
Calcium‐dependent inactivation of heteromeric NMDA receptor‐channels expressed in human embryonic kidney cells.
Authors: Medina I , Filippova N , Charton G , Rougeole S , Ben-Ari Y , Khrestchatisky M , Bregestovski P
Published: December 2014

1. Whole‐cell current through heteromeric NR1‐NR2A and NR1‐NR2B subunit combinations of NMDA channels transiently expressed in human embryonic kidney cells (HEK 293) were studied using the patch‐clamp technique. 2. With 4 mM Mg‐ATP in the internal pipette solution, the responses of cells expressing NR1‐NR2A channels to glutamate application gradually decreased, reaching 50% of control during the first 20 min of recording. This process was accompanied by acceleration of desensitization. 3. Conditioning (5‐15 s) applications of glutamate (100 microM) induced a transient inactivation of NR1‐NR2A and NR1‐NR2B channels (20‐40%) with a slow time course of recovery (tau r = 10‐60 s). Both the degree of inactivation and the time constant of recovery increased with the duration of conditioning applications of glutamate, and with an elevation of Ca2+ in the external solution. 4. These results show that both NR1‐NR2A and NR1‐NR2B recombinant NMDA receptor‐channels expressed in HEK 293 cells can be transiently inhibited by Ca2+ ions in a similar way to that described for hippocampal neurones.
Respiratory phase resetting and airflow changes induced by swallowing in humans.
Authors: Paydarfar D , Gilbert R J , Poppel C S , Nassab P F
Published: December 2014

1. Relationships between the timing of respiration and deglutition were studied in thirty awake healthy subjects at rest. Deglutition was monitored by submental electromyography, pharyngeal manometry and videofluoroscopy. Respiration was recorded by measurement of oronasal airflow and chest wall movement. Three types of deglutition were studied: injected bolus swallows, spontaneous swallows, and visually cued swallows of boluses previously placed in the mouth. 2. The effect of each swallow on respiratory rhythm was characterized by measurement of cophase, defined as the interval between the onset of deglutitive submental EMG activity to the onset of subsequent rescheduled inspirations. Cophase was determined for swallows initiated at different phases of the respiratory cycle. In all subjects deglutition caused phase resetting of respiratory rhythm. Cophase was largest for swallows initiated near the the inspiratory‐expiratory (E‐I) transition and smallest for swallows initiated near the expiratory‐inspiratory (E‐I) transition. The pattern of respiratory resetting by deglutition was topologically classified as type 0. This pattern was shown for swallows induced by bolus injection or visual cue, and for spontaneous swallows. 3. The incidence of spontaneous deglutition was influenced by the position of the swallow in the respiratory cycle. Few spontaneous swallows were initiated near the E‐I transition whereas most occurred from late inspiration to mid‐expiration. 4. Deglutition caused an abrupt decrease in airflow leading to an interval of apnoea, followed by a period of expiration. The duration of deglutition apnoea for spontaneous swallows was shorter than that for 5 ml bolus swallows, and was unaffected by the respiratory phase of swallow initiation. The period of expiration after swallowing was longest for swallows initiated at the I‐E transition, and shortest for E‐I swallows. 5. The intervals between bolus injection and the onset of deglutition apnoea, and the timing of swallowing events, were not significantly altered by the phase in the respiratory cycle at which swallowing was exhibited. 6. To quantify the relationship between bolus flow and respiration, we determined the latencies between cessation of inspiratory airflow and arrival of the bolus at the larynx (alpha), and between laryngeal bolus departure and resumption of inspiratory airflow (delta). Both values were dependent upon the respiratory phase of swallowing. The lowest values for alpha and delta were found for early‐inspiratory and late‐expiratory swallows, respectively. 7. We conclude that swallowing causes respiratory phase resetting with a pattern that is characteristic of the strong perturbations of an attractor‐cycle oscillator.(ABSTRACT TRUNCATED AT 400 WORDS)
Gramicidin‐perforated patch recording: GABA response in mammalian neurones with intact intracellular chloride.
Authors: Ebihara S , Shirato K , Harata N , Akaike N
Published: December 2014

1. By the development of a new perforated patch method using gramicidin, the effects of GABA on neurones dissociated from the rat substantia nigra pars reticulata (SNR) were examined without disturbing the intracellular chloride concentration. 2. Using the patch pipette solution containing gramicidin (100 micrograms ml‐1), the access resistance dropped to less than 20 M omega within 40 min after making the gigaohm seal. 3. Under current‐clamp conditions, GABA caused a hyperpolarization accompanied by a blockade of spontaneous firing. Under voltage clamp at a holding potential (Vh) of ‐50 mV, GABA evoked an outward current by way of bicuculline‐ and picrotoxin‐sensitive GABAA receptors. 4. A 10‐fold change of extracellular chloride concentration resulted in a 58 mV shift of the reversal potential of GABA‐induced outward current (EGABA), indicating that the membrane behaves like a chloride electrode in the presence of GABA. 5. The intracellular chloride activities (aCli), calculated with the Nernst equation using both extracellular chloride activity and EGABA values, ranged from 2.8 to 19.7 mM with a mean value of 9.5 mM. The aCli was not affected either by different pipette solutions or by different holding potentials more hyperpolarized than ‐40 mV. 6. In the recording from SNR neurones in brain slice using the gramicidin‐perforated patch‐clamp technique, the inhibitory and excitatory postsynaptic currents were recorded in different current directions and the former was blocked by bicuculline. 7. In conclusion, the gramicidin‐perforated patch method will disclose previously unknown aspects of biological responses involving Cl‐.