bluuebunny/crossref_metadata_2025 huggingface dataset:
Necrosis and apoptosis associated with distinct Ca2+ response patterns in target cells attacked by human natural killer cells.
Authors: Oshimi Y , Oshimi K , Miyazaki S
Published: December 2014

1. During the process of cell death, rises in cytosolic Ca2+ concentration ([Ca2+]i) together with structural changes were investigated in target cells attacked by purified CD3‐,CD16+ human natural killer (NK) cells. 2. In the target cell line K562, a rapid [Ca2+]i rise to 1‐2 microM occurred a few minutes after NK cell‐target cell contact, immediately followed by leakage of the Ca2+ indicator dye fura‐2 from the cell. Cells were permeabilized, but their chromatin was not fragmented. The changes were basically consistent with those seen in necrosis induced by activated complement. 3. In the target cell line MOLT‐4, which expressed the apoptosis‐inducing surface antigen Fas much more strongly than K562, the majority of attacked cells displayed a [Ca2+]i rise to 0.7‐1 microM followed by a slow decline, often associated with diminishing [Ca2+]i oscillations. As a whole, [Ca2+]i remained higher than 150 nM for at least 1.5‐3 h (approximately 100 nM in control cells). 4. MOLT‐4 cells attacked by NK cells became bubble shaped within 20 min of the main [Ca2+]i rise reaching its peak, and then both the cell and chromatin were fragmented into small pieces. These findings were basically consistent with those in apoptosis induced by a monoclonal antibody against the surface antigen Fas. 5. NK cells induced both necrosis and apoptosis in cell lines insensitive to NK cells in the presence of an antibody against the major histocompatibility complex class I (antibody‐dependent cell‐mediated cytotoxicity, ADCC). The distinct Ca2+ responses patterns described above corresponded to necrosis or apoptosis in different cells stimulated by the common ADCC pathway. 6. Human NK cells were found to be capable of inducing necrosis (membrane damage) or apoptosis (nuclear damage) depending on the target cell types. The characteristic Ca2+ response profile was a good indicator for distinguishing between the modes of cell death induced by the cytotoxicity of the killer cells.
Potentiation of IPSCs by nitric oxide in immature rat sympathetic preganglionic neurones in vitro.
Authors: Wu S Y , Dun N J
Published: December 2014

1. Whole‐cell patch‐clamp recording techniques were applied to sympathetic preganglionic neurones (SPNs), the majority of which contain neuronal nitric oxide synthase (NOS), in transverse thoracolumbar spinal cord slices from 8‐ to 12‐day‐old rats, and the role of nitric oxide (NO) in modulating the inhibitory postsynaptic current (IPSC) evoked by focal stimulation was examined. 2. Superfusing the slices with the NO precursor L‐arginine (L‐Arg, 300 microM) and the NO donor sodium nitroprusside (SNP, 100 microM) potentiated IPSCs from several minutes to more than 1 h; the increase was prevented by pretreating the slices with bovine haemoglobin (100 microM), the nitric oxide synthase inhibitor N omega‐nor‐L‐arginine (NO‐Arg, 100 microM) or the soluble guanylate cyclase inhibitor 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ, 1 microM). Haemoglobin, NO‐Arg and ODQ slightly reduced the IPSCs evoked in some of the SPNs. 3. Superfusion of the slices with N2,2'‐O‐dibutyrylguanosine 3',5'‐cyclic monophosphate (dbcGMP, 300 microM) in the presence of the phosphodiesterase inhibitor 3‐isobutyl‐1‐methylxanthine (IBMX, 750 microM) reversibly increased the IPSCs as well. 4. While enhancing the IPSCs, L‐Arg or SNP had no significant effects on outward currents induced by pressure application of the putative inhibitory transmitter glycine in the same cells. 5. A train of suprathreshold depolarizing current pulses (30 Hz for 10 s) injected into the recording SPNs caused, after a delay of several minutes, a long‐lasting increase of IPSCs; this effect was nullified by superfusing the slices with haemoglobin or NO‐Arg. 6. The result suggests that NO released endogenously from SPNs may act as a retrograde messenger molecule to enhance the release of glycine or a related substance, possibly via a cGMP‐dependent mechanism.
Attenuation of the hypoxic ventilatory response in adult rats following one month of perinatal hyperoxia.
Authors: Ling L , Olson E B , Vidruk E H , Mitchell G S
Published: December 2014

1. This study was designed to test the hypothesis that perinatal suppression of peripheral arterial chemoreceptor inputs attenuates the hypoxic ventilatory response in adult rats. Perinatal suppression of peripheral chemoreceptor activity was achieved by exposing rats to hyperoxia throughout the first month of life. 2. Late‐gestation pregnant rats were housed in a 60% O2 environment, exposing the pups to hyperoxia from several days prior to birth until they were returned to normoxia on postnatal day 28. These perinatally treated rats were then reared to adulthood (3‐5 months old) in normoxia. In addition to the mother rats, adult male rats were also exposed to hyperoxia, creating an adult‐treated control group. Two to four months after the hyperoxic exposure, treated rats were compared with untreated male rats of similar age. 3. A whole‐body, flow‐through plethysmograph was used to measure hypoxic and hypercapnic ventilatory responses of the unanaesthetized adult rats. In moderate hypoxia (arterial oxygen partial pressure, Pa,O2 approximately 48 mmHg). VE (minute ventilation) and the ratio VE/VCO2 (ventilation relative to CO2 production) increased by 16.7 +/‐ 4.0 and 35.4 +/‐ 3.4%, respectively, in perinatal‐treated rats (means +/‐ S.E.M.), but increased more in untreated control rats (51.4 +/‐ 2.8 and 83.1 +/‐ 4.3%; both P < 10(‐6)). 4. In contrast to the impaired hypoxic ventilatory response, ventilatory responses to hypercapnia (5% CO2) were similar between untreated control and perinatal‐treated rats. 5. Impaired hypoxic responsiveness was unique to the perinatal‐treated rats since hypoxic ventilatory responses were not attenuated in adult‐treated rats. 6. The results indicate that ventilatory responses to hypoxaemia are greatly attenuated in adult rats that had experienced hyperoxia during their first month of life, and suggest that normal hypoxic ventilatory control mechanisms are susceptible to developmental plasticity.
Effects of cardiac glycosides on excitation‐contraction coupling in frog skeletal muscle fibres.
Authors: Sárközi S , Szentesi P , Jona I , Csernoch L
Published: December 2014

1. The effects of digoxin and ouabain on the calcium release flux from the sarcoplasmic reticulum (SR), isometric tension and intramembrane charge movement were studied in voltage clamped skeletal muscle fibres of the frog. 2. Both cardiac glycosides increased both calcium transients and simultaneously recorded tension at all membrane potentials, showing different effects on the peak and on the steady components of the calcium release flux. These effects were attained at an extracellular digoxin concentration of 5 nM and an estimated intracellular ouabain concentration of 1‐2 nM. Digoxin and ouabain thus exerted their effects at the same concentration on calcium release in skeletal muscle as previously observed in isolated cardiac‐type ryanodine receptor (RyR) calcium release channels. 3. The peak of SR calcium release increased at all voltages, with the largest potentiation at intermediate membrane potentials. This increase in calcium release flux was attained despite an unchanged SR calcium content. The attenuated release rate therefore reflected an increased number of open RyR channels rather than increased SR loading. 4. These effects could be attributed to an increase in calcium release activation and not a decrease in the rate of inactivation. Rather, the rate of inactivation was enhanced at all voltages as expected from the increased calcium concentration in the triadic junction. 5. In contrast, CMA (17 alpha‐acetoxy‐6‐chloro‐4, 6‐pregnadiene‐3,20‐dione; 5 microM), a Na(+)‐K(+)‐ATPase inhibitor with no positive inotropic effects on the heart, neither influenced SR calcium release nor antagonized the effects of ouabain. 6. Both digoxin and ouabain preserved total intramembrane charge apart from a small negative shift in the mid‐point voltage and increase in slope factor. 7. Both digoxin and ouabain induced calcium release from heavy SR vesicles at rates comparable to that induced by ryanodine or caffeine. 8. It is concluded that at least part of the inactivating component of SR calcium release involves distinct RyR calcium release channels that resemble the cardiac RyR isoform in its specific sensitivity to cardiac glycosides.
Lasting effects of glutamate on nuclear calcium concentration in cultured rat hippocampal neurons: regulation by calcium stores.
Authors: Korkotian E , Segal M
Published: December 2014

1. Changes in free intracellular calcium concentrations ([Ca2+]i) were measured in the nucleus and perinuclear regions of cultured rat hippocampal neurons using either fura‐2 or fluo‐3 calcium indicators. 2. Brief application of glutamate caused a transient rise of [Ca2+]i in all cell compartments, which recovered to pre‐drug levels in all but the nuclear region. The new, higher level of nuclear calcium ([Ca2+]n) was sustained for as long as the cell was monitored. 3. The new level of [Ca2+]n was dependent on the magnitude of the calcium transient, and was higher in older cells in culture, but it did not affect responses to subsequent applications of glutamate. 4. The sustained elevation of [Ca2+]n was prevented by drugs which affect calcium stores (caffeine, ryanodine and Ruthenium Red), indicating that an extranuclear calcium store interacts with [Ca2+]n.
Ca(2+)‐ and GTP‐dependent exocytosis in mouse pancreatic beta‐cells involves both common and distinct steps.
Authors: Proks P , Eliasson L , Ammälä C , Rorsman P , Ashcroft F M
Published: December 2014

1. The effects of GTP and Ca2+ on secretion from single pancreatic beta‐cells were studied using capacitance measurements as an indicator of exocytosis. 2. GTP or GTP gamma S produced a concentration‐dependent increase in cell capacitance in the absence of intracellular calcium. There was no effect of cyclic AMP or BAPTA an GTP‐induced secretion. 3. In the absence of GTP, the relationship between intracellular calcium concentration and the maximum rate of secretion was fitted by the Hill equation with a slope factor of 2.5 and half‐maximal activation at 1.6 microM intracellular Ca2+. Similar values were obtained in the presence of GTP gamma S, suggesting GTP does not alter the sensitivity of the secretory machinery to Ca2+. 4. GDP beta S alone had no effect on cell capacitance but caused a dose‐dependent inhibition of exocytosis induced by infusion of either GTP gamma S or Ca2+, suggesting both stimuli involve G‐protein activation. GDP beta S was without effect on exocytosis evoked by depolarization‐mediated Ca2+ entry. 5. The time course of exocytosis following rapid elevation of GTP gamma S by photolysis of a caged precursor was dependent on the intracellular Ca2+ and cyclic AMP concentrations. 6. Our results are interpreted in terms of a model in which the secretory pathways stimulated by Ca2+ and GTP contain both common and separate parts.
Induction and maintenance of ganglionic long‐term potentiation require activation of 5‐hydroxytryptamine (5‐HT3) receptors.
Authors: Alkadhi K A , Salgado-Commissariat D , Hogan Y H , Akpaudo S B
Published: December 2014

1. An extracellular recording technique was used to study the effects of 5‐hydroxytryptamine (5‐HT, serotonin) on the tetanus‐induced long‐term potentiation (LTP) of the nicotinic pathway of transmission in the superior cervical ganglion (SCG) of the rat. The postganglionic compound action potential (CAP), made submaximal by treatment with hexamethonium (O.4 mM), was used as an index of transmission in the ganglion. 2. Serotonin (10 microM) markedly enhanced the magnitude of LTP without affecting the post‐tetanic potentiation (PTP). The serotonin (2‐30 microM) concentration‐response curve for LTP was bell shaped as no enhancement was seen with 30 microM serotonin. This may largely be due to activation of a 5‐HT1 receptor subtype and not to desensitization. 3. When superfused before tetanus, the 5‐HT1A receptor agonist 8‐hydroxydipropylamino‐tetralin (8‐OH‐DPAT, 5 microM) prevented the expression of LTP without affecting PTP. 4. Pretreatment of ganglia with the 5‐HT2 receptor agonist R‐(+)‐dimethoxy‐4‐iodoamphetamine (R‐(+)‐DOI, 1 microM) enhanced the tetanus‐induced LTP. Similar treatment with the 5‐HT2 receptor antagonist ketanserin (3 microM) had no significant effect on LTP. 5. Pretreatment of ganglia with the 5‐HT3 receptor agonist 1‐m‐(chlorophenyl) biguanide (m‐CPGB, 1 microM), markedly increased (300%) the tetanus‐induced LTP. Similar pretreatment with the 5‐HT3 receptor antagonist 3‐tropanyl‐3,5‐dichlorobenzoate (MDL 72222, 0.5 microM) completely prevented the expression of LTP. Fully expressed LTP was reversibly blocked by MDL 72222 when applied during the maintenance phase of LTP. 6. Tetanic stimulation of monoamine‐depleted ganglia (from reserpine‐pretreated rats, 3 mg kg‐1 for 24 h) failed to induced LTP. 7. In monoamine‐depleted ganglia, tetanus preceded by superfusion with m‐CPBG readily induced LTP. MDL 72222 completely blocked this LTP. However in these ganglia tetanus failed to induced LTP when m‐CPBG was given 2 min (during PTP) or 1 h after tetanus. 8. Tetanic stimulation of monoamine‐depleted ganglia in the presence of R‐(+)‐DOI failed to induced LTP. 9. We conclude that tetanus‐induced LTP of the SCG of the rat requires activation of 5‐HT3 receptors both for induction and maintenance.
Myosin heavy chain isoform expression and high energy phosphate content in human muscle fibres at rest and post‐exercise.
Authors: Sant'Ana Pereira J A , Sargeant A J , Rademaker A C , de Haan A , van Mechelen W
Published: December 2014

1. The relationship between myosin heavy chain (MyHC) isoforms and high energy phosphate content was studied in human muscle fibres at rest and following maximal dynamic exercise lasting 25 s. 2. Single fibre fragments were characterized as type I, type IIA, type IIX or type IIAX. These latter fibres were subdivided into five groups on the basis of the proportion of MyHC IIX isoform present. 3. Resting ATP concentration in type I fibres was 10% lower than in type II fibres (P < 0.05), but no differences were found amongst type IIA, IIX and IIAX fibre groups. Phosphocreatine (PCr) content was lower in type I than in type II fibres (P < 0.01) and, amongst type II fibres, increased progressively with the amounts of MyHC IIX expressed. 4. After 25 s of maximal dynamic exercise ATP concentration was reduced in all fibres. The decrease was approximately 25% in type I fibres and between 47 and 66% in the type II subgroups. 5. Post‐exercise PCr content was low in all fibre types. Fibre groups with the lowest post‐exercise PCr also had the lowest ATP and the highest inosine monophosphate contents. delta PCr (rest to post‐exercise) was smallest in type I fibres and showed a progressive increase in the type II fibre groups as the proportion of the faster IIX myosin heavy chain isoform increased.
A large‐conductance K+ channel that is inhibited by the cytoskeleton in the smooth muscle cell line DDT1 MF‐2.
Authors: Ehrhardt A G , Frankish N , Isenberg G
Published: December 2014

1. DDT1 MF‐2 smooth muscle cells responded to the bath application of histamine or ATP with an increase in the cytosolic Ca2+ concentration ([Ca2+]c) and the whole‐cell K+ current, IK(BA). 2. In cell‐attached patches, histamine (100 microM) activated currents through a 200 pS K+ channel (‘BKA’ channel). In the absence of agonists, the BKA channel was activated by excision of the patch. Both histamine and patch excision increased the channel activity (NPo; where N is the number of channels per patch and Po is the open probability) by reducing the long closures between the bursts of openings. 3. In inside‐out patches, the BKA channel had a conductance of 201 +/‐ 4 pS (symmetrical solutions of 150 mM KCl, 2 mM MgCl2 and 2 mM EGTA). Replacement of K+ in the patch electrode by Na+, Li+ or Cs+ prevented the flow of inward currents and reduced the outward K+ conductance to 113 pS. 4. NPo was insensitive to changes in [Ca2+]c from 10 nM to 1 microM. NPo was also not modified either by cytosolic Na+, ATP, GTP, GTP gamma S, dithiothreitol or TEA (10 mM) or by extracellular 4‐aminopyridine (5 mM), glibenclamide (20 microM) or TEA (10 mM). The BKA channel was blocked by 5 mM intracellular BaCl2 or by 10 nM extracellular iberiotoxin. 5. In cell‐attached patches, BKA channel activity could be induced by 1 microM cytochalasin B, applied either through the patch pipette or in the bath solution. The effects of cytochalasin B, of patch excision, or of histamine on NPo were not additive but saturative. 6. Whole DDT1 MF‐2 cells had resting potentials of ‐10 mV, dominated by the chloride conductance; the resting potential changed to ‐82 mV when the K+ conductance was increased by cytochalasin B or by histamine. The effects of cytochalasin B and histamine on IK(BA) were not additive but saturative. 7. We discuss the hypothesis that the interaction between the cytoskeleton and the BKA channel promotes the long channel closures; depolymerization of F‐actin may constitute a mechanism by which the agonists histamine or ATP disinhibit BKA channel activity.
Interactions between the histidine stimulation of cadmium and zinc influx into human erythrocytes.
Authors: Horn N M , Thomas A L
Published: December 2014

1. Histidine (2‐40 mM) stimulated cadmium uptake into human erythrocytes incubated in the presence of 1% bovine serum albumin to ensure that the free, ionic cadmium concentration was low. 2. The histidine‐stimulated cadmium uptake correlated with the calculated concentration of the cadmium‐bis‐histidine complex rather than the cadmium‐mono‐histidine complex or free ionic cadmium. 3. The histidine stimulation of cadmium uptake was saturable and stereospecific. D‐Histidine (10 mM) had no effect. 4. Cadmium and zinc were both able to inhibit 65Zn2+ uptake into erythrocytes incubated in the presence of 40 mM L‐histidine. The relationships between the percentage inhibition of 65Zn2+ uptake and the calculated concentrations of cadmium‐bis‐histidine and zinc‐bis‐histidine were very similar, which suggests that the metal histidine complexes compete for a common transport mechanism. 5. Pretreatment of the erythrocytes with N‐ethylmaleimide using a protocol which is known to inhibit the system y+ amino acid transport mechanism had no effect on the histidine stimulation of metal transport.
Activation of oxytocin neurones by systemic cholecystokinin is unchanged by morphine dependence or withdrawal excitation in the rat.
Authors: Brown C H , Munro G , Murphy N P , Leng G , Russell J A
Published: December 2014

1. Morphine inhibits supraoptic nucleus oxytocin neurones directly and presynaptically via inhibition of afferent noradrenergic endings. 2. We studied whether morphine tolerance/dependence (induced by intracerebroventricular (I.C.V.) morphine infusion) alters the responsiveness of oxytocin neurones to systemic cholecystokinin (CCK), a stimulus which activates oxytocin neurones via the release of noradrenaline. 3. CCK (20 micrograms kg‐1, i.v.) increased plasma oxytocin concentrations similarly in urethane‐anaesthetized morphine‐naive and ‐dependent rats. In naive rats, I.C.V. (10 micrograms) and i.v. morphine (0.5 mg kg‐1) reduced CCK‐induced oxytocin secretion by 95 +/‐ 4 and 49 +/‐ 10%, respectively. In dependent rats, i.v. morphine reduced CCK‐induced release by only 8 +/‐ 9%, indicating tolerance. 4. In urethane‐anaesthetized rats, i.v. CCK increased the firing rates of oxytocin neurones similarly in morphine‐naive and ‐dependent rats (by 1.2 +/‐ 0.2 and 1.4 +/‐ 0.3 spikes s‐1 maximum, respectively, over 5 min). Naloxone did not alter spontaneous or CCK‐induced activity in naive rats but increased activity in dependent rats (by 3.4 +/‐ 0.5 spikes s‐1), indicative of withdrawal excitation; however, the response to CCK remained unchanged after naloxone. 5. Systemic CCK did not trigger withdrawal, nor did it have a greater excitatory effect in dependent rats. Thus, morphine withdrawal excitation of oxytocin neurones does not involve supersensitivity to the noradrenergic input, or hypersensitivity of this input to i.v. CCK. Tolerance apparently occurs both at the cell bodies of oxytocin neurones in the supraoptic nucleus and in their noradrenergic input. However, dependence is apparent only at the cell bodies.
Late depression of muscle excitability in humans after fatiguing stimulation.
Authors: McFadden L K , McComas A J
Published: December 2014

1. Changes in muscle excitation and in isometric twitch force have been studied for up to 8 h after fatiguing stimulation of the human biceps brachii. 2. Within 10 s of a cessation of the 20 Hz fatiguing tetanus, the amplitudes of the M waves (muscle compound action potentials) had returned to control values, whereas the twitch forces were reduced in all subjects. The M waves then decreased in amplitude over the next 3 h, reaching a mean value that was 42.4 +/‐ 18.6% of control levels (means +/‐ S.E.M.; P < 0.001). 3. By 8 h, the mean M wave amplitude had recovered to 93.8 +/‐ 33.3% of control levels, while the corresponding mean twitch force was 104.1 +/‐ 36.9%. 4. The cellular mechanism responsible for the depression of the M wave is presently unknown, but it is likely to be postsynaptic and may involve Na+ channels.
Anion secretion induced by capacitative Ca2+ entry through apical and basolateral membranes of cultured equine sweat gland epithelium.
Authors: Ko W H , Chan H C , Wong P Y
Published: December 2014

1. Anion secretion induced by capacitative Ca2+ entry through apical and basolateral membranes of cultured equine sweat gland epithelium was studied using the short‐circuit current (Isc) technique. 2. Thapsigargin induced an increase in Isc that could be inhibited when external Ca2+ was chelated by EGTA. 3. The inhibition of the thapsigargin‐induced Isc could be reversed by re‐addition of Ca2+ to apical or basolateral solutions. The magnitude of the reactivated Isc depended predominantly on basolateral Ca2+ concentration. 4. The magnitude of the reactivated Isc upon basolateral Ca2+ addition increased with the thapsigargin concentration, indicating its dependence on the emptied state of the Ca2+ store induced by thapsigargin. 5. The thapsigargin‐induced Isc, as well as the Ca(2+)‐dependent reactivation of Isc in EGTA‐treated epithelia, was inhibitable by apical, but not basolateral, addition of flufenamate, and by basolateral addition of La3+. Other Ca2+ channel blockers, verapamil and nifedipine, had no effect when applied to either membrane. 6. The results suggest that thapsigargin‐induced anion secretion by the equine sweat gland epithelial cells is crucially dependent upon the Ca2+ influx occurring primarily through the basolateral membrane, and that apical and basolateral membranes may possess different pathways for Ca2+ entry.
Neurones in the ventrolateral pons are required for post‐hypoxic frequency decline in rats.
Authors: Coles S K , Dick T E
Published: December 2014

1. The breathing pattern following acute hypoxia (arterial O2 pressure (Pa,O2), 27.4 +/‐ 7.7 mmHg) was measured in intact, anaesthetized and spontaneously breathing adult rats (n = 4) and in anaesthetized, vagotomized, paralysed and ventilated animals (n = 14). Measurements were made both before and after bilateral lesions or chemical inactivation of neurones in the lateral pons. Respiratory motor activity was recorded as an index of the respiratory cycle. We tested the hypothesis that the ventrolateral pons is required for expression of post‐hypoxic frequency decline, defined as a decrease in respiratory frequency below steady‐state baseline levels following brief exposures to hypoxia. 2. We identified an area in the ventrolateral pons where brief (1 ms) low current (< or = 20 microA) pulses evoked a short‐latency inhibitor of phrenic nerve activity. At this site, bilateral electrical or chemical lesions (n = 3) were performed, or neural activity was inhibited by focal injections of 10 mM muscimol (n = 9). In six control animals, neural activity was inhibited by muscimol injections into the lateral pons, dorsal to the target site. 3. Prior to pontine intervention, respiratory frequency decreased below baseline levels following 20‐110 s of 8% O2. The decrease in frequency resulted from a prolongation of expiration (up to 276%), which gradually returned to baseline levels (tau = 45 s). 4. Following lesions or inhibition of neural activity in the ventrolateral pons, baseline inspiratory (TI) and expiratory (TE) durations were altered, albeit minimally, in the animals with intact vagus nerves. Expiratory duration following hypoxia was not different from baseline levels either in vagotomized (P = 0.18) or intact (P > 0.05) animals. In contrast, injections of muscimol at more dorsal sites did not alter the decrease in frequency normally seen following hypoxia. 5. Histological examination revealed that effective lesion or injection sites were within the lateral pontine tegmental field and included portions of the noradrenergic A5 cell group. 6. We conclude that the mechanism responsible for post‐hypoxic frequency decline involves an active neural process that depends on the integrity of the ventrolateral pons.
Laminar origins of inhibitory synaptic inputs to pyramidal neurons of the rat neocortex.
Authors: Nicoll A , Kim H G , Connors B W
Published: December 2014

1. Inhibitory neuron‐pyramidal cell interactions were investigated in slices of rat somatosensory cortex in which excitatory synaptic transmission was blocked with bath‐applied glutamate receptor antagonists. Local inhibitory neurons were excited by focal pressure ejections of small (approximately 40 pl) volumes of 1‐10 mM acetylcholine. 2. The frequency of inhibitory postsynaptic potentials (IPSPs) ("responses per trial' or R/T) declined as the stimulation distance was increased. Inhibitory inputs were most prevalent in layer II/III regular spiking (RS) pyramidal neurons (30 cells) where median R/T was 0.020. In layer V, the median R/T was 0.024 for RS neurons (25 cells), but significantly lower for burst‐firing (IB) neurons (17 cells), where median R/T was 0.007 (P = 0.039). 3. IPSPs in individual layer V pyramidal cells were recorded with CsCl electrodes. In eight neurons, spontaneous picrotoxin‐sensitive IPSPs were recorded and found to display a wide range of 10‐90% rise times (1‐34 ms), not correlated with amplitude (0.2‐18 mV). For a further ten pyramidal neurons, extracellular stimulating electrodes were placed simultaneously in layers II/III and V/VI in order to evoke pairs of IPSPs whose waveforms were averaged and compared. In seven cells, IPSPs evoked from layer II/III (distal location) had longer 10‐90% rise times than IPSPs evoked from layer V/VI stimulating electrodes (proximal location). In addition, "proximal' IPSPs could always be reversed by membrane depolarization whereas "distal' ones could not (n = 4/4). 4. This study showed that pyramid cell‐inhibitory neuron interconnections are extensive but their spatial organization varies with cell class and with cortical layer. In addition, pyramidal neurons can receive inhibitory inputs from locations on their apical dendrites.
Motor units of juvenile rat lumbrical muscles and fibre type compositions of the glycogen‐depleted component.
Authors: Ridge R M , Rowlerson A
Published: December 2014

1. Fourth deep lumbrical muscles were dissected out, with their nerve supply, from juvenile rats aged 8‐15 days (a period corresponding to maximal rate of decline of polyneuronal innervation), and aged 28, 29 and 30 days (when developmental synapse elimination is complete). Preparations were superfused with rat Ringer solution at 25 degrees C. 2. Isometric twitches and tetani were recorded from the whole muscles and from a single motor unit in the muscle. Unit isolation was by partial section of the sural or lateral plantar nerves. The axon of a single unit occurred naturally in the sural nerve in some cases. 3. Fibres in single units were depleted of glycogen by repetitive stimulation, and studied histologically in frozen midbelly sections of the muscle, stained for glycogen with periodic acid‐Schiff's reagent (PAS). Most fibre counts were based on transmittance measurements made with an image analysis system. Contralateral muscles were unstimulated and acted as controls. 4. Motor unit sizes were estimated from tetanic tensions and from muscle fibre cross‐sectional area measurements. Comparison of the two methods indicated that in most units glycogen depletion was not complete. This effect was maximal at 8 and 10 days postnatally. It is suggested that this is due to weak neuromuscular transmission at synapses in the process of natural elimination during development. 5. Other sections (serial and semi‐serial) were immunostained with a polyclonal antibody raised against slow myosin. Fibres staining for the antibody (slow; S‐fibres) contribute about 12‐9% of muscle fibres depending on age. In some muscles, fibre types were determined by myosin ATPase staining following alkali pre‐incubation. Fast fibres (F‐fibres) contained no slow myosin. 6. Some units had no S‐fibres (i.e. they were homogeneous), and many units had a small proportion of S‐fibres, though less than in the whole muscle (i.e. they were heterogeneous but composition was biased in favour of F‐fibres). 7. One unit from a 10‐day‐old rat contained more S‐ than F‐fibres. Many of the F‐fibres were small. It is proposed that this was a developing IIC/IIA unit, a type known to occur in adults. 8. It is concluded that mismatched connections in developing motor units possibly become weak early (by 8 days postnatally) in the process of synapse elimination (which is complete by 20 days postnatally), but that the time course of actual withdrawal cannot be followed by the technique of glycogen depletion.
Postsynaptic glutamate uptake in rat cerebellar Purkinje cells.
Authors: Takahashi M , Sarantis M , Attwell D
Published: December 2014

1. Whole‐cell clamp experiments on Purkinje neurons in rat cerebellar slices were used to test whether glutamate transporters, detected immunocytochemically in the somata and dendrites of the cells, are functional in the cell surface membrane, and to investigate their role in terminating synaptic transmission. 2. A membrane current was detected with the pharmacology, voltage and ion dependence of a glutamate uptake current. Part of the current was generated by an anion conductance activated when uptake occurs. 3. With sodium and glutamate inside the cell, raising the external potassium concentration generated an outward current attributable to reversed operation of glutamate transporters. 4. The magnitude of the uptake current suggested that Purkinje cell transporters could help to terminate transmission at the climbing and parallel fibre to Purkinje cell synapses. Reducing postsynaptic glutamate uptake with intracellular D‐aspartate prolonged the climbing fibre EPSC. 5. These data establish the existence of functional postsynaptic glutamate transporters, show that they contribute to terminating synaptic transmission, and suggest that they may play a role in the preferential death of Purkinje cells in ischaemia.
C‐polymodal nociceptors activated by noxious low temperature in human skin.
Authors: Campero M , Serra J , Ochoa J L
Published: December 2014

1. Fifty‐five C‐polymodal nociceptors innervating hairy skin in human volunteers were tested for discrete stimulus‐response properties through microneurography. 2. All fifty‐five units were responsive to mechanical and heat stimuli. Twenty‐two (40%) of these exhibited an additional response to noxious low temperature. The twenty‐two mechano‐heat‐cold nociceptors displayed similar receptor responses to heat and mechanical stimuli, and conduction velocity, as those of the pure mechano‐heat C‐polymodal nociceptors. 3. Low temperature stimuli between 19 and 0 degree C evoked responses at low discharge frequency (0.4 +/‐ 0.22 impulses s‐1, mean +/‐ S.E.M.) in the twenty‐two units sensitive to such energy. These units displayed a tendency to decrease their discharge after a few seconds of steady stimulation. 4. Three units tested with a freezing stimulus responded with relatively vigorous discharge, which never exceeded the maximal discharge frequency elicited by either mechanical or heat stimuli. One of these units became sensitized to heat after the freezing challenge. 5. It is concluded that a subpopulation of C‐polymodal nociceptors is sensitive to noxious low temperature and may thus contribute to the determination of cold pain. The same type of primary afferent is probably one mediator of the symptom cold hyperalgesia in peripheral nerve disease.
Optical imaging of spatiotemporal patterns of glutamatergic excitation and GABAergic inhibition in the guinea‐pig auditory cortex in vivo.
Authors: Horikawa J , Hosokawa Y , Kubota M , Nasu M , Taniguchi I
Published: December 2014

1. Glutamatergic excitation and gamma‐aminobutyric acid (GABA)‐ergic inhibition in layers II and III of the auditory cortex of anaesthetized guinea‐pigs were recorded optically using a voltage‐sensitive dye RH795 and a 12 x 12 photodiode array. 2. After contralateral ear stimulation with pure tones, transient excitatory responses followed by inhibitory responses were observed in fields A (primary) and DC of the auditory cortex. The area of the excitatory responses was sandwiched or surrounded by the areas of the inhibitory responses. 3. Optically recorded excitatory responses to pure tones had two components: a component sensitive to 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX), a non‐N‐methyl‐D‐aspartate (non‐NMDA) receptor antagonist, and a component sensitive to 2‐amino‐5‐phosphono‐valerate (APV), an NMDA receptor antagonist. Application of CNQX (5 microM) to the auditory cortex suppressed an early, but not a late, phase of the excitation; application of APV (100 microM) had the opposite effect. Concomitant application of CNQX and bicuculline methiodide (BMI, 4 microM), a GABAA receptor antagonist, increased the amplitude of the late phase 4‐fold. This enhanced response was suppressed by APV. 4. These results indicate that (i) auditory cortical excitatory responses are mediated by both non‐NMDA and NMDA receptors, (ii) inhibition is mediated by GABAA receptors, (ii) the excitatory bands are sandwiched or surrounded by GABAA receptor‐mediated inhibitory areas and (iv) GABAA receptors effectively inhibit the NMDA, but not the non‐NMDA, receptor‐mediated excitation.
Purinoceptor‐operated cationic channels in human B lymphocytes.
Authors: Markwardt F , Löhn M , Böhm T , Klapperstück M
Published: December 2014

1. Using the patch clamp method in the outside‐out configuration, purinoceptor‐dependent unitary currents were measured in tonsillar and transformed tonsillar human B lymphocytes. 2. Single channel currents were evoked by ATP4‐, the free‐acid form of ATP, and by 2',3' O‐benzoyl‐4‐benzoyl‐ATP (BzATP) in the micromolar concentration range, but not by 10 mM ADP3‐ or 0.5 mM Mg(2+)‐bound ATP. 3. The channels could be activated and deactivated several times for as long as 30 min even in the absence of intracellular ATP, GTP, or glucose. 4. The channels were selective for small cations and had a conductance of 9 pS with Cs+ as the intracellular and Na+ as the extracellular monovalent cation. 5. The half‐maximal activation of the channels was obtained by 114 microM ATP4‐ and by 16 microM BzATP. The increase in the open probability after raising the ATP4‐ concentration was mainly due to a decrease in the times the channels spend in the closed state. 6. It is concluded that human B lymphocytes possess cationic channels directly gated by extracellular ATP4‐. Their agonist binding characteristics are typical for P2z purinoceptors, but their permeation behaviour is different from the large non‐specific pores formed by ATP4‐ in fibroblasts, macrophages and mast cells.
Latent addition in motor and sensory fibres of human peripheral nerve.
Authors: Bostock H , Rothwell J C
Published: December 2014

1. The time constants of motor and sensory nerve fibres were studied in normal human ulnar nerves by the method of latent addition, using threshold tracking to follow the recovery of excitability after brief conditioning current pulses. The 60 microseconds test and conditioning stimuli were applied at the wrist, and the conditioning stimuli were set to 90, 60, 30, ‐30, ‐60 and ‐90% of the control threshold current. Compound muscle action potentials were recorded from abductor digiti minimi, and sensory nerve action potentials from the little finger. 2. Recovery from depolarizing conditioning pulses was slower than recovery from hyperpolarizing pulses and strongly dependent on conditioning pulse amplitude. The voltage dependence of latent addition was attributed to subthreshold activation of sodium channels (local response). 3. Motor and sensory nerve excitability generally recovered from ‐90% hyperpolarizing pulses as the sum of two exponential components, although the slow component was negligible in some motor nerves. The fast component (time constant 43.3 +/‐ 2.0 microseconds, mean +/‐ S.E.M., n = 9) was similar between motor and sensory fibres in the same subject. It showed no consistent voltage dependence, and was attributed to a passive input time constant of the fibres. The slow component of recovery from hyperpolarizing pulses was greater in sensory than in motor fibres and was voltage dependent: it could be greatly increased in motor and sensory fibres by steady depolarization. It was attributed to a regenerative membrane current, active at the resting potential in sensory and at least some motor nerves. 4. The latent addition responses were compared with the computed responses of four theoretical models. Both motor and sensory responses were well fitted by a model in which a fraction of the sodium channels (less in motor than in sensory fibres) were activated at potentials 20 mV more negative than normal and at half the normal rate, and did not inactivate. 5. It is concluded that the differences in latent addition between motor and sensory fibres are primarily due to differences in non‐classical, voltage‐dependent ion channels, active close to the resting potential. These "threshold channels' may help to account for the longer strength‐duration time constant of sensory fibres, for their lower rheobase, and for their greater tendency to fire repetitively.
Regulation of an outwardly rectifying Cl‐ conductance in single proximal tubule cells isolated from frog kidney.
Authors: Robson L , Hunter M
Published: December 2014

1. A previous study has identified a Cl‐ conductance (GCl) in single proximal tubule cells isolated from frog kidney, which was activated by a protein kinase C (PKC)‐dependent mechanism. 2. The whole‐cell patch clamp technique was employed to examine further the properties and regulation of GCl. 3. GCl showed outward rectification, outward conductance was significantly greater than the inward conductance (56.1 +/‐ 15.6 vs. 16.8 +/‐ 6.4 microS cm‐2, respectively, n = 8). DIDS (4,4'‐diisothiocyanatostilbene‐2,2'‐disulphonic acid) blocked GCl in a dose‐ and voltage‐dependent manner. 4. Other anions permeated the conductance. The anion selectivity sequence, I‐ > Br‐ > Cl‐ > gluconate, followed Eisenman's sequence I. 5. GCl could be activated by ATP. This process was dependent on ATP hydrolysis and channel phosphorylation. 6. G‐protein activation inhibited the ATP‐dependent activation of GCl. 7. These data support the hypothesis that activation of GCl by an increase in cell volume is dependent on ATP hydrolysis and channel phosphorylation via PKC.
Two types of low‐voltage‐activated Ca2+ channels in neurones of rat laterodorsal thalamic nucleus.
Authors: Tarasenko A N , Kostyuk P G , Eremin A V , Isaev D S
Published: December 2014

1. The pharmacological and kinetic properties of two types of low‐voltage‐activated (LVA) Ca2+ currents were studied in thalamocortical neurones of the laterodorsal (LD) thalamic nucleus during early postnatal development. The whole‐cell patch‐clamp technique was used on brain slices from rats of three age groups: 12, 14 and 17 days old (postnatal day (P) 12, P14 and P17). 2. In P12 neurones, the population of LVA Ca2+ channels was homogeneous. LVA Ca2+ current elicited by depolarizing voltage steps from a holding potential more negative than ‐70 mV was sensitive to nifedipine (Kd = 2.6 microM). This current reached a maximum at about ‐55 mV and had a fast monoexponential decay with a time constant, tau h,f, of 32.3 +/‐ 4.0 ms. 3. The population of LVA Ca2+ channels in P14 and P17 neurones was found to be heterogeneous. A subpopulation of nifedipine‐insensitive LVA Ca2+ channels was observed. The current‐voltage curve of the Ca2+ current had a characteristic hump with two peaks at about ‐65 and ‐55 mV. As well as the fast component (designated IT,f), the decay of the LVA current also included a slow component (designated IT,s), with inactivation time constants (tau h,s) of 54.2 +/‐ 4.5 and 68.6 +/‐ 3.17 ms for P14 and P17 neurones, respectively. 4. The kinetics of both components could be well approximated by the m2h Hodgkin‐Huxley equation. No significant difference in activation kinetics was observed. The activation time constants for the fast (tau m,f) and slow (tau m,s) components were 6.3 +/‐ 1.0 and 7.3 +/‐ 1.5 ms, respectively. 5. La3+ at a concentration of 1 microM effectively blocked the IT,f component but Ni2+ (25 microM) completely eliminated the IT,s component. 6. Steady‐state inactivation curves of both components could be best fitted by a Boltzmann function with membrane potential values at half‐maximal inactivation of ‐85.5 and ‐98.1 mV for the fast and slow components, respectively. 7. It was concluded that two different subtypes of LVA Ca2+ channel are present in LD neurones. Only the fast type is well expressed at the earliest postnatal stage (P12). The slow type could be found at the end of the second week (P14). The amplitude of the slow current increased progressively up to P17, obviously coinciding with dendritic expansion as judged by progressive increase of the membrane capacitance of the corresponding neurones. This property appears to differentiate neurones of the associative nuclei from neurones of other thalamic nuclei.
Chronic hypoxia suppresses pharmacomechanical coupling of the uterine artery in near‐term pregnant sheep.
Authors: Hu X Q , Zhang L
Published: December 2014

1. The role of inositol 1,4,5‐trisphosphate (InsP3) in the reduced vascular responsiveness to 5‐hydroxytryptamine (5‐HT) caused by chronic hypoxia was examined in uterine arteries obtained from normoxic (control) and chronically hypoxic pregnant sheep (approximately 140 days gestation) maintained at high altitude (3820 m; arterial PO2, 60 mmHg) from 30 days gestation. 2. Chronic hypoxia significantly decreased uterine artery contractile sensitivity in that pD2 (‐logEC50) for the contractile response to 5‐HT was 7.19 +/‐ 0.15 and 6.62 +/‐ 0.12 (P < 0.05) in uterine arteries from normoxic and chronically hypoxic sheep, respectively. The intrinsic efficacy of the agonist was reduced by 75%. Although 5‐HT2A receptor density (Bmax) in the uterine artery was not changed in chronically hypoxic sheep compared with normoxic sheep (32.0 +/‐ 9.8 vs. 31.9 +/‐ 5.9 fmol (mg protein)‐1, respectively) as assessed from the saturation binding of [3H]ketanserin, the agonist binding affinity (pKA, ‐log of dissociation constant) was decreased from 6.25 +/‐ 0.07 in normoxic sheep to 5.85 +/‐ 0.08 in chronically hypoxic sheep (P < 0.05). 3. Chronic hypoxia did not change the time course of 5‐HT‐induced InsP3 synthesis but decreased its potency in inducing InsP3 synthesis, with the pD2 being 6.09 +/‐ 0.11 and 5.51 +/‐ 0.08 (P < 0.05) in uterine arteries from normoxic and chronically hypoxic sheep, respectively. The maximal response of 5‐HT‐induced InsP3 generation in the uterine artery was decreased from 251.3 +/‐ 24.2 pmol (mg protein)‐1 in normoxic sheep to 146.6 +/‐ 11.3 pmol (mg protein)‐1 in chronically hypoxic sheep (P < 0.05). Furthermore, the ability of the activated 5‐HT receptors to couple InsP3 synthesis was significantly decreased in chronically hypoxic compared with normoxic sheep (280 +/‐ 10 vs. 450 +/‐ 20 fmol InsP3 (fmol receptor)‐1, P < 0.01). In addition, for a given amount of InsP3 generated, the contractile force of the uterine artery was significantly less in chronically hypoxic sheep (0.82 +/‐ 0.08 g tension (pmol InsP3)‐1) than that in normoxic sheep (1.28 +/‐ 0.05 g tension (pmol InsP3)‐1) (P < 0.05). 4. These results suggest that chronic hypoxia suppresses pharmacomechanical coupling of the ovine uterine artery by inhibiting the efficiency of receptor‐effector‐contraction coupling. This suppression of the InsP3 pathway may play an important role in the adjustment of vascular tone and uterine blood flow in response to the stress of chronic hypoxia in late pregnancy.
Local synaptic release of glutamate from neurons in the rat hypothalamic arcuate nucleus.
Authors: Belousov A B , van den Pol A N
Published: December 2014

1. The hypothalamic arcuate nucleus (ARC) contains neuroendocrine neurons that regulate endocrine secretions by releasing substances which control anterior pituitary hormonal release into the portal blood stream. Many neuroactive substances have been identified in the ARC, but the existence of excitatory neurons in the ARC and the identity of an excitatory transmitter have not been investigated physiologically. 2. In the present experiments using whole‐cell current‐ and voltage‐clamp recording of neurons from cultures and slices of the ARC, we demonstrate for the first time that some of the neurons in the ARC secrete glutamate as their transmitter. 3. Using microdrop stimulation of presynaptic neurons in ARC slices, we found that local axons from these glutamatergic neurons make local synaptic contact with other neurons in the ARC and that all evoked excitatory postsynaptic potentials could be blocked by the selective ionotropic glutamate receptor antagonists 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX; 10 microM) and D,L‐2‐amino‐5‐phosphonovalerate (AP5; 100 microM). To determine the identity of ARC neurons postsynaptic to local glutamatergic neurons, we used antidromic stimulation to reveal that many of these cells were neuroendocrine neurons by virtue of their maintaining axon terminals in the median eminence. 4. In ARC cultures, postsynaptic potentials, both excitatory and inhibitory, were virtually eliminated by the glutamate receptor antagonists AP5 and CNQX, underlining the functional importance of glutamate within this part of the neuroendocrine brain. 5. GABA was secreted by a subset of ARC neurons from local axons. The GABAA receptor antagonist bicuculline released glutamatergic neurons from chronic inhibition mediated by synaptically released GABA, resulting in further depolarization and an increase in the amplitude and frequency of glutamate‐mediated excitatory postsynaptic potentials.