bluuebunny/crossref_metadata_2025 huggingface dataset:
Albumin stimulates uptake of calcium into subcellular stores in rat cortical astrocytes.
Authors: Nadal A , Fuentes E , McNaughton P A
Published: December 2014

1. When albumin from either plasma or serum is applied at low concentrations to cortical astrocytes a decrease in the level of [Ca2+]i is observed. At higher concentrations trains of calcium spikes are seen. 2. Removal of the polar lipids which are normally bound to native albumin abolishes the ability to induce spikes, but the decrease in [Ca2+]i is unaffected. The decrease is abolished by the denaturation of albumin and is not reproduced by a number of other proteins, and is therefore a specific action of albumin. We conclude that native albumin has a dual agonist action: the decrease in [Ca2+]i is induced by the albumin protein molecule, while the spikes are induced by a lipid normally bound to it. 3. The decrease is rapid (fastest tau = 12 s) and the rate is dependent on the concentration of albumin. [Ca2+]i falls from 77 nM to around 34 nM in the presence of saturating levels of albumin, and this level appears to be maintained indefinitely. 4. The decrease is due to an uptake of calcium into subcellular stores, as it is not abolished by removal of external Ca2+ or Na+ but is abolished by thapsigargin and cyclopiazonic acid, which are specific inhibitors of the endoplasmic reticulum Ca(2+)‐ATPase. 5. When the state of store filling after albumin application is probed with a pulse of glutamate it can be seen that stores fill with the same time course as the decrease in [Ca2+]i. The low level of [Ca2+]i in albumin must therefore be maintained by a suppression of calcium influx rather than by a continued uptake into stores. 6. The calcium uptake potentiates the efficacy of low concentrations of calcium‐releasing agonists such as glutamate and bradykinin by almost an order of magnitude. 7. A possible function for the calcium uptake caused by albumin is to potentiate the production of calcium spike trains by promoting refilling of calcium stores in the intervals between spikes. The uptake may play a role in the response of astrocytes to damage in the CNS.
Central motor command activates sympathetic outflow to the cutaneous circulation in humans.
Authors: Vissing S F , Hjortsø E M
Published: December 2014

1. The aim of this study was to provide direct evidence that sympathetic outflow to the skin in humans is governed by central neural mechanisms. 2. Microneurographic measurements of skin sympathetic nerve activity (SNA) from the personal nerve was performed in nine subjects during: (1) static hand grip at 10, 20 and 30% maximal voluntary contraction (MVC); and (2) attempted static hand grip during partial neuromuscular blockade produced by injection of vecuronium. 3. Two minutes of static hand grip at 20 and 30% MVC (force output, 9.6 +/‐ 0.2 and 14.4 +/‐ 0.3 kg, respectively) evoked significant increase in skin SNA that were graded to the intensity of the exercise. Static hand grip at 10% MVC (force output, 4.8 +/‐ 0.1 kg) caused a small but insignificant increase in skin SNA. 4. During vecuronium‐induced neuromuscular blockade, subjects failed to maintain a force output equivalent to the output produced during 10% MVC before vecuronium (force output: 1st min, 4.4 +/‐ 0.6 kg; 2nd min, 2.1 +/‐ 0.4 kg), in spite of maximal effort being applied. This attempted hand grip exercise consistently evoked considerable increase in skin SNA that did not significantly differ from the responses produced by hand grip at 30% MVC; total skin SNA increased by 246 +/‐ 93% during 2 min of attempted hand grip and increased by 243 +/‐ 77% during 2 min of static hand grip at 30% MVC (means +/‐ S.E.M., P < or = 0.05). These increase in skin SNA were not due to activation of resting muscles because measurements of surface electromyography showed no activity in resting forearm muscles during static or attempted hand grip exercise. 5. This study provides direct neurophysiological evidence that central motor command can activate sympathetic outflow. During static hand grip, central motor command is the primary mechanism that stimulates sympathetic outflow to skin.
Slow inactivation of Na+ current and slow cumulative spike adaptation in mouse and guinea‐pig neocortical neurones in slices.
Authors: Fleidervish I A , Friedman A , Gutnick M J
Published: December 2014

1. Spike adaptation of neocortical pyramidal neurones was studied with sharp electrode recordings in slices of guinea‐pig parietal cortex and whole‐cell patch recordings of mouse somatosensory cortex. Repetitive intracellular stimulation with 1 s depolarizing pulses delivered at intervals of < 5 s caused slow, cumulative adaptation of spike firing, which was not associated with a change in resting conductance, and which persisted when Co2+ replaced Ca2+ in the bathing medium. 2. Development of slow cumulative adaptation was associated with a gradual decrease in maximal rates of rise of action potentials, a slowing in the post‐spike depolarization towards threshold, and a positive shift in the threshold voltage for the next spike in the train; maximal spike repolarization rates and after‐hyperpolarizations were unchanged. 3. The data suggested that slow adaptation reflects use‐dependent removal of Na+ channels from the available pool by an inactivation process which is much slower than fast, Hodgkin‐Huxley‐type inactivation. 4. We therefore studied the properties of Na+ channels in layer II‐III mouse neocortical cells using the cell‐attached configuration of the patch‐in‐slice technique. These had a slope conductance of 18 +/‐ 1 pS and an extrapolated reversal potential of 127 +/‐ 6 mV above resting potential (Vr) (mean +/‐ S.E.M.; n = 5). Vr was estimated at ‐72 +/‐ 3 mV (n = 8), based on the voltage dependence of the steady‐state inactivation (h infinity) curve. 5. Slow inactivation (SI) of Na+ channels had a mono‐exponential onset with tau on between 0.86 and 2.33 s (n = 3). Steady‐state SI was half‐maximal at ‐43.8 mV and had a slope of 14.4 mV (e‐fold)‐1. Recovery from a 2 s conditioning pulse was bi‐exponential and voltage dependent; the slow time constant ranged between 0.45 and 2.5 s at voltages between‐128 and ‐68 mV. 6. The experimentally determined parameters of SI were adequate to simulate slow cumulative adaptation of spike firing in a single‐compartment computer model. 7. Persistent Na+ current, which was recorded in whole‐cell configuration during slow voltage ramps (35 mV s‐1), also underwent pronounced SI, which was apparent when the ramp was preceded by a prolonged depolarizing pulse.
Cutaneous inhibitory receptive fields of withdrawal reflexes in the decerebrate spinal rat.
Authors: Weng H R , Schouenborg J
Published: December 2014

1. The inhibitory cutaneous input to the withdrawal reflex pathways to single hindlimb muscles was investigated in decerebrate spinal rats (n = 53) using electromyography. 2. Withdrawal reflexes in the peronei, extensor digitorum longus and tibialis anterior muscles of the leg were strongly inhibited by conditioning mechanical, thermal (CO2 laser) and intracutaneous electrical stimulation of specific skin areas. By contrast, withdrawal reflexes in the biceps posterior‐semitendinosus muscles of the thigh could only be weakly inhibited by conditioning skin stimulation. 3. Powerful inhibition of withdrawal reflexes in single lower leg muscles was elicited from the ipsilateral hindpaw plantar area, which would move towards the stimulation on contraction in the respective muscle. In addition, weak nociceptive inhibition was evoked from the corresponding skin areas on the contralateral hindlimb and, in some muscles, the tail. 4. The ipsilateral inhibitory and excitatory receptive fields of the withdrawal reflexes in single muscles overlapped somewhat. On stimulation of these transitional areas the reflex responses were preceded by a short‐lasting inhibition. 5. Graded mechanical and thermal stimulation demonstrated prominent inhibitors effects from nociceptive receptors. Weak inhibitory effects were elicited by innocuous mechanical stimulation, suggesting a weak contribution from low threshold mechanoreceptors. Latency measurements indicated an inhibitory input from both myelinated and unmyelinated fibres. 6. In conclusion, the withdrawal reflex pathways receive a powerful nociceptive inhibitory input through spinal pathways. The movement‐related organization of this input suggests that it serves to prevent inappropriate withdrawal reflexes.
Calcium‐dependent prepotentials contribute to spontaneous activity in rat tuberomammillary neurons.
Authors: Stevens D R , Haas H L
Published: December 2014

1. Intracellular recordings from histaminergic neurons of the tuberomammillary (TM) nucleus reveal subthreshold depolarizing potentials (DPs) which persist in the presence of tetrodotoxin. 2. Block of hyperpolarization‐activated current by 1‐4 mM Cs+ failed to reduce spontaneous activity or DPs. 3. In the presence of tetrodotoxin DPs are voltage dependent and are depressed by Cd2+ and Co2+. 4. Ba2+ (100 microM) treatment enhances DP amplitude and converts low‐amplitude potentials to tetrodotoxin‐insensitive action potentials. 5. In the presence of TTX, DPs are reduced by Ni2+. Spontaneous action potentials are also reduced by Ni2+ (100‐300 microM). A low‐threshold Ca2+ current is present which is sensitive to Ni2+. These results indicate the presence of calcium currents, perhaps of the low‐threshold type, which contribute to activation of action potentials in TM neurons.
Extracellular potassium and chemosensitivity in the rat carotid body, in vitro.
Authors: Pepper D R , Landauer R C , Kumar P
Published: December 2014

1. The effects of raising extracellular potassium concentration ([K+]o) from 3.0 to 5.3, 9.5 or 16.8 mM on chemoreceptor responses to hypoxia, hypercapnia and asphyxia were examined in a superfused in vitro rat carotid body preparation. 2. Single‐exponential functions with offset were fitted to the chemoreceptor discharge responses to ramp decreases in Po2. Increasing [K+]o was without effect upon the rate constants of the fitted exponential functions (P > 0.20). Increasing [K+]o, significantly increased the horizontal asymptote (chemoreceptor discharge in hyperoxia) in a non‐linear fashion when all levels of [K+]o were included in the analysis (P < 0.001) but not when a comparison was made only between 3.0 and 5.3 mM Ko+ (P > 0.40). The rightward position of the response curves, as quantified by the Po2 at 50% maximum discharge, was linearly related to [K+]o but only when all levels of [K+]o were included in the analysis (P < 0.03). Chemoreceptor sensitivity to [K+]o increased non‐linearly as [K+]o was increased but this effect was not dependent upon the Po2 (P > 0.90). 4. Increasing PCO2 in hyperoxia increased chemoreceptor discharge linearly at all levels of [K+]o. Whilst discharge at any level of PCO2 was elevated by increased levels of [K+]o, raising [K+]o did not increase CO2 sensitivity (P > 0.20). Similarly, increasing PCO2 did not increase chemosensitivity to [K+]o. The lack of effect of [K+]o upon CO2 chemosensitivity was also observed as Po2 was decreased to hypoxic levels (P > 0.10). 5. Our data demonstrate that an elevation of [K+]o can increase chemoreceptor discharge in the in vitro carotid body in a PO2‐ and PCO2‐independent manner, suggesting that the PO2‐dependent effects of [K+]o, previously reported in vivo may be due to other indirect effects of [K+]o or hypoxia.
Ca2+ responses to ATP via purinoceptors in the early embryonic chick retina.
Authors: Sugioka M , Fukuda Y , Yamashita M
Published: December 2014

1. The action of adenosine triphosphate on cytoplasmic Ca2+ concentration ([Ca2+]i) was studied in the retinal cell of early embryonic chicks with fura‐2 fluorescence measurements. The fluorescence was measured from the whole neural retina dissected from chick embryos at embryonic day three (E3). 2. Bath application of ATP (> or = 30 microM; EC50, 128 microM) raised [Ca2+]i by the release of Ca2+ from intracellular Ca2+ stores, since the Ca2+ response to ATP occurred even in a Ca(2+)‐free medium. 3. The Ca2+ response to ATP was mediated by P2U purinoceptors. An agonist for P2U purinoceptors, uridine triphosphate (UTP), evoked Ca2+ rises more potently (> or = 3 microM; EC50, 24 microM) than ATP. Agonists for P2X purinoceptors, alpha, beta‐methylene ATP and beta, gamma‐methylene ATP, or an agonist for P2Y purinoceptors, 2‐methylthio ATP (500 microM each), caused no Ca2+ response. Suramin (100 microM) and Reactive Blue 2 (50 microM) almost completely blocked the Ca2+, responses to 500 microM ATP and 200 microM UTP. 4. The developmental profile of the Ca2+ response to ATP was studied from E3 to E13. The Ca2+ response to ATP was largest at E3, drastically declined towards E8 and decreased further until E11‐13. 5. These results suggest that the Ca2+ mobilization by ATP via P2U purinoceptors is characteristic of early embryonic retinal cells.
Different types of Na+ and A‐type K+ currents in dorsal root ganglion neurones innervating the rat urinary bladder.
Authors: Yoshimura N , White G , Weight F F , de Groat W C
Published: December 2014

1. Whole‐cell patch‐clamp recording in combination with axonal tracing techniques was used to examine the electrical properties of afferent neurones innervating the urinary bladder of the adult rat. Individual bladder afferent cells were labelled by Fast Blue (FB), injected into the bladder wall. 2. Passive and active electrical parameters at room temperature (20‐22 degrees C) in FB‐labelled bladder afferent neurones were comparable with those in unlabelled neurones. Unselected dorsal root ganglion (DRG) neurones as well as bladder afferent neurones exhibited two different types of action potential: high‐threshold humped spikes in small‐sized neurones and low‐threshold narrow spikes in large‐sized neurones. 3. The majority (70%) of bladder neurones which were small in size expressed high‐threshold tetrodotoxin (TTX)‐resistant Na+ channels and slow‐inactivating A‐type K+ channels (KA), which were available at the resting membrane potential, whereas large‐sized DRG neurones had low‐threshold TTX‐sensitive Na+ channels and fast‐inactivating KA channels, which were almost completely inactivated at the resting membrane potential. 4. Half‐maximal conductances of activation of TTX‐resistant and TTX‐sensitive Na+ currents were obtained at ‐10.3 and ‐25.3 mV, respectively. The TTX‐resistant and TTX‐sensitive Na+ currents were half‐inactivated at ‐25.3 and ‐56 mV, respectively. 5. In the TTX‐resistant neurones, the transient outward K+ current (A‐type current, IA) with half‐maximal conductance at ‐40.8 mV was half‐inactivated at ‐77.5 mV, and exhibited slower decaying kinetics (mean decay constant (tau), 240 ms) than the IA current recorded from the large‐sized TTX‐sensitive neurones (mean tau, 20 ms). 6. These results suggest that the majority of bladder afferent neurones have high electrical thresholds for spike activation due to the TTX‐resistant Na+ current and the slow‐inactivating IA current, which reflect the large population of unmyelinated high‐threshold C fibre afferents that innervate the urinary bladder.
Mechanisms of long‐lasting hyperpolarizations underlying slow sleep oscillations in cat corticothalamic networks.
Authors: Contreras D , Timofeev I , Steriade M
Published: December 2014

1. To explore the nature of the long‐lasting hyperpolarizations that characterize slow oscillations in corticothalamic circuits in vivo, intracellular recordings were obtained under ketamine‐xylazine anaesthesia from cortical (Cx) cells of the cat precruciate motor cortex, thalamic reticular (RE) cells from the rostrolateral sector, and thalamocortical (TC) cells from the ventrolateral (VL) nucleus. 2. Measurements in the three cell types showed input resistance (Rin) to be highest during the long‐lasting hyperpolarizations that correspond to depth‐positive waves of the cortical EEG. Rin was lowest during the early phase of high‐amplitude depth‐negative EEG waves and increased thereafter until the next cycle of the slow oscillation. 3. Spontaneous long‐lasting hyperpolarizations were compared with those evoked by dorsal thalamic stimulation. Voltage versus current (V‐I) plots showed similar membrane potential (Vm) ranges and slopes for spontaneous and evoked hyperpolarizations in both Cx and RE cells. V‐I plots from TC cells had similar slopes, but Vm during evoked hyperpolarizations was displaced towards more negative values. 4. Intracellular injection of constant hyperpolarizing current in Cx cells increased the amplitude of the initial part of the depolarizing plateau of the slow oscillation, but decreased the amplitude of the last part. 5. These results suggest disfacilitation to be the dominant mechanism in the membrane of cortical and thalamic cells during the spontaneous long‐lasting hyperpolarizations, which shape and synchronize slow oscillations in corticothalamic networks. In Cx and RE cells, the same mechanism underlies thalamically evoked long‐lasting hyperpolarizations. By contrast, evoked responses in TC cells show a strong additional hyperpolarizing factor. We propose that GABAB processes are stronger in TC than in Cx neurones, thus rendering the thalamus an easier target for absence‐type epileptic phenomena through potentiation of thalamic rebound capabilities.
The effect of acetylcholine on finger capillary pressure and capillary flow in healthy volunteers.
Authors: Morris S J , Kunzek S , Shore A C
Published: December 2014

1. Constitutive nitric oxide (NO) synthase has been demonstrated in human skin microvascular endothelial cells; however, the physiological significance of this finding is not known. The aim of this study was to investigate the effects of acetylcholine (ACh), which stimulates the release of NO from endothelial cells, on skin capillary pressure, capillary pulse pressure amplitude (CPPA) and capillary red blood cell velocity (CBV) in healthy volunteers. 2. Finger nailfold capillary pressure was measured in five healthy volunteers. CBV was measured in capillaries of the dorsal middle phalangeal area of the finger in six subjects using a recently developed capillary anemometer. In each case the responses to ionophoretically applied ACh and vehicle were measured on two separate fingers on the left hand. 3. Application of vehicle did not significantly change either capillary pressure, CPPA or CBV. ACh significantly increased capillary pressure (from 15.8 +/‐ 2.2 mmHg under basal conditions to 27.7 +/‐ 3.8 mmHg at the plateau of the ACh response; P < 0.008), CPPA (from 2.4 +/‐ 2.4 mmHg at baseline to 8.4 +/‐ 2.4 mmHg at the plateau of the drug response; P < 0.013) and CBV (from 0.54 +/‐ 0.22 mm s‐1 at baseline to 2.46 +/‐ 1.12 mm s‐1 after ACh; P < 0.008). 4. The increases in capillary pressure, CPPA and CBV following the application of ACh suggest that the overall effect of ACh was to induce a reduction in the pre‐ to postcapillary resistance ratio.
Bicarbonate transport in sheep parotid secretory cells.
Authors: Steward M C , Poronnik P , Cook D I
Published: December 2014

1. Intracellular pH (pH1) was measured by microfluorimetry in secretory endpieces isolated from sheep parotid glands and loaded with the pH‐sensitive fluoroprobe 2', 7'‐bis(2‐carboxyethyl)‐5(6)‐carboxyfluorescein (BCECF). 2. Stimulation with 1 microM acetylcholine (ACh) caused a large, transient decrease in pH1 of 0.37 +/‐ 0.02 pH units followed by a slower recovery. The transient, which was reduced by 60% in the absence of HCO3‐, could be attributed mainly to HCO3‐ efflux. During sustained stimulation, pH1 increased to a value that exceeded the resting value by 0.083 +/‐ 0.023 pH units after 20 min. 3. The anion channel blocker NPPB (0.1 mM) reduced the transient acidification in response to ACh by 48% and raised pH1 during sustained stimulation. Simultaneous application of NPPB and ACh accelerated the re‐alkalinization following the initial acidification, indicating that NPPB inhibits HCO3‐ efflux. 4. The stilbene derivative H2DIDS (0.5 mM) reduced the transient acidification in response to ACh by 76% but caused a marked decrease in pH1 during sustained stimulation. Simultaneous application of H2DIDS and ACh slowed the re‐alkalinization following the initial acidification, indicating that the main effect of H2DIDS was to inhibit HCO3‐ accumulation. 5. In the absence of HCO3‐, the recovery from an acid load was unaffected by ACh stimulation. Acid extrusion, although dependent on Na+, was not inhibited by amiloride (1 mM), clonidine (1 mM) or H2DIDS (0.5 mM) and was therefore provisionally attributed to a Na(+)‐H+ exchanger isoform other than NHE1 or NHE2. 6. In the presence of HCO3‐, the rate of recovery from an acid load was reduced during ACh stimulation, probably as a result of the increased efflux of HCO3‐. Acid extrusion was dependent on Na+ and was significantly inhibited by H2DIDS. 7. We conclude that ACh‐evoked HCO3‐ secretion in the sheep parotid gland differs from that in many other salivary glands by being driven predominantly by basolateral Na(+)‐HCO3‐ cotransport rather than by Na(+)‐H+ exchange.
Respiratory mechanics during exhaustive submaximal exercise at high altitude in healthy humans.
Authors: Cibella F , Cuttitta G , Kayser B , Narici M , Romano S , Saibene F
Published: December 2014

1. The present investigation was conducted to test the hypothesis that the respiratory system is stressed more during exhaustive exercise in chronic hypoxia than in normoxia. 2. Four healthy male subjects (aged 33‐35 years) exercised on a cycle ergometer at 75% of the local maximum oxygen consumption (Vo2,max) until exhaustion, at sea level (SL) and after a 1 month stay at 5050 m (HA). 3. Airflow at the mouth (V), oesophageal (Po) and gastric (Pg) pressures were measured at rest, during exercise and recovery. Minute ventilation (VE), respiratory power (Wresp), respiratory frequency (f) and transdiaphragmatic pressure (Pdi) were calculated from the measured variables. 4. The subjects' mechanical power output of cycling at HA was 23.7% lower than at SL. In spite of this reduction, time to exhaustion at HA was 55.3% less than at SL. VE increased slightly during exercise at SL, but showed a marked increase at HA, and at the end of exercise at HA was 47.3% higher than at SL. 5. Respiratory power increased more at HA than at SL (77.3% higher at the end of exercise) due to the increase in f needed to sustain the high VE. 6. Gastric pressure swings were negative at the end of HA exercise but always positive at SL. The Pai:Po ratio reached values below 1 at HA but never at SL. 7. These data seem to indicate that the respiratory system is stressed more during submaximal exercise at HA than at SL. We suggest that the exceedingly high VE demand, requiring an excessive Wresp, may lead to fatigue of the diaphragm.
Red nucleus lesions abolish the biphasic respiratory response to isocapnic hypoxia in decerebrate young rabbits.
Authors: Waites B A , Ackland G L , Noble R , Hanson M A
Published: December 2014

1. The respiratory response to isocapnic hypoxia (inspired O2 fraction (FI,O1), 0.1‐0.12) was measured in twelve vagotomized, paralysed, artificially ventilated young rabbits (aged 26.6 +/‐ 0.4 days), following pre‐collicular decerebration. Phrenic nerve efferent activity was used as an index of central respiratory output (RO). In hypoxia RO increased after 1‐2 min (phase 1) but decreased over the subsequent 3‐4 min to, or below, the pre‐hypoxic control level (phase 2). 2. We used electrical stimulation to target areas in the mesencephalon which inhibit RO. Profiles of the response to stimulation were determined in a grid of electrode penetrations made mediolaterally and rostrocaudally at the level of the superior colliculi, in normoxia. Histology confirmed that stimulation in the red nucleus (RN) inhibited RO profoundly. 3. Electrolytic lesions were made bilaterally in RN inhibitory sites or in adjacent areas. The respiratory response to isocapnic hypoxia was measured again post‐lesioning. 4. In six rabbits with bilateral lesions in the RN, phase 2 of the respiratory response was abolished and RO remained elevated throughout the hypoxic exposure. However, in six rabbits with unilateral lesions in the RN, or with bilateral lesions placed in areas outside the RN that did not inhibit RO on electrical stimulation, the respiratory response remained biphasic. 5. In both groups of animals, blood pressure increased during 1‐3 min of hypoxia before decreasing to pre‐hypoxic levels. This cardiovascular response remained biphasic irrespective of whether animals showed a biphasic respiratory response or a sustained increase in RO after lesioning. 6. We conclude that structures within the RN are crucial to the mechanism producing a fall in RO during isocapnic hypoxaemia in the neonate.
ADP‐induced rapid inward currents through Ca(2+)‐permeable cation channels in mouse, rat and guinea‐pig megakaryocytes: a patch‐clamp study.
Authors: Kawa K
Published: December 2014

1. The rapid inward currents in mouse megakaryocytes evoked by adenosine diphosphate (ADP), a ubiquitous platelet‐activating substance, were studied. Time and current resolution were improved by using patch‐clamp recording and an extracellular fast perfusion ("Y tube') technique. 2. Application of ADP (40 microM) to megakaryocytes immersed in physiological saline evoked rapid inward currents (80‐340 pA at ‐42 mV). The cellular responses to a second ADP application were markedly reduced, but in the absence of external Ca2+, responses to repeated ADP application were maintained and did not deteriorate. 3. The ADP‐induced current recorded in Ca(2+)‐free external media showed short latency (less than 20 ms) and approximately exponential decay (time constant, 300‐500 ms), which was independent of the holding potential and seemed to be caused mainly by receptor desensitization; it took over 5.5 min for complete recovery. 4. The ADP concentration response relationship of the megakaryocytes revealed that the half‐maximal concentration and the Hill coefficient were 12.6 microM and 1.4, respectively. 5. An ion replacement experiment showed that the ADP‐induced currents could be carried by Na+, Cs+ and K+, but not Cl‐, and the cation channels were permeable to Ca2+, Ba2+ and Mg2+. 6. Neither Ca2+ chelators (10 mM EGTA and 10 mM BAPTA) nor hydrolysis‐resistant guanine nucleotides (2 mM GDP‐beta‐S and 0.4 mM 5'‐guanylylimidodiphosphate) in the internal saline affected the rapid responses to ADP, and ADP‐induced currents were recorded in excised membrane patches, suggesting that the ADP receptor site and the molecular structure forming the cation channel are tightly coupled and/or parts of the same molecule. 7. In rat and guinea‐pig megakaryocytes, ADP‐induced rapid inward currents showed the same properties as in mouse megakaryocytes.
A HERG‐like K+ channel in rat F‐11 DRG cell line: pharmacological identification and biophysical characterization.
Authors: Faravelli L , Arcangeli A , Olivotto M , Wanke E
Published: December 2014

1. The relationships between the K+ inward rectifier current present in neuroblastoma cells (IIR) and the current encoded by the human ether‐á‐go‐go‐related gene (HERG), IHERG, and the rapidly activating repolarizing cardiac current IK(r), were investigated in a rat dorsal root ganglion (DRG) x mouse neuroblastoma hybrid cell line (F‐11) using pharmacological and biophysical treatments. 2. IIR shared the pharmacological features described for IK(r), including the sensitivity to the antiarrhythmic drugs E4301 and WAY‐123,398, whilst responding to Cs+, Ba2+ and La3+ in a similar way to IHERG. 3. The voltage‐dependent gating properties of IIR were similar to those of IK(r) and IHERG, although IIR outward currents were negligible in comparison. 4. In high K+ extracellular solutions devoid of divalent cations, IIR deactivation kinetics were removed resulting in long‐lasting currents apparently activated in hyperpolarization, with a marked (2.7‐fold) increase in conductance, as recorded from the instantaneous linear current‐voltage relationship at ‐120 mV. Re‐addition of Ca2+ restored the original closure of the channel whereas re‐addition of Mg2+ reduced the peak current. 5. The IIR described here, the heart IK(r) and the IHERG could be successfully predicted by a unique kinetic model where the voltage dependencies of the activation/inactivation gates were properly voltage shifted. On the whole, IIR seems to be the first example of a HERG‐type current constitutively expressed and operating in mammalian cells of the neuronal lineage.
Absence of Ca2+ current facilitation in skeletal muscle of transgenic mice lacking the type 1 ryanodine receptor.
Authors: Fleig A , Takeshima H , Penner R
Published: December 2014

1. Whole‐cell patch‐clamp recordings were used to study voltage‐dependent facilitation of Ca2+ currents and excessive Ca2+ tail current in skeletal myoballs cultured from wild‐type and transgenic mice expressing a null mutation of the ryanodine receptor (RyR) type 1 (dyspedic myoballs). 2. Ca2+ current density in dyspedic myoballs was reduced by about 60% compared with wild‐type cells, with dihydropyridine‐binding capacity largely retained. 3. Strong and long‐lasting depolarizations (+80 mV and 600 ms), which normally produce excessive tail currents upon repolarization in control cells, failed to do so in dyspedic myoballs. 4. Dyspedic myoballs also failed to produce both Ca2+ current facilitation and the left shift of the current‐voltage (I‐V) curve induced by paired‐pulse stimulation. 5. We propose that excessive tail currents and facilitation arise from silent Ca2+ channels acting as the voltage sensors in excitation‐contraction coupling.
Calcium currents, transmitter release and facilitation of release at voltage‐clamped crayfish nerve terminals.
Authors: Wright S N , Brodwick M S , Bittner G D
Published: December 2014

1. The presynaptic terminals at crayfish (Procambarus spp.) opener neuromuscular junctions were voltage clamped. Calcium currents were measured during (ICa) and following (tail ICa) presynaptic depolarizations; EPSPs or IPSPs were simultaneously recorded from the (postsynaptic) muscle fibre directly beneath the presynaptic impalement. 2. For short (< or = 6 ms) presynaptic depolarizations, most of the transmitter release occurred during the tail ICa. EPSP or IPSP amplitudes at the end of the 6 ms pulse (end EPSP or end IPSP) increased monotonically with the integral of the ICa ([symbol: see text]ICa). The suppression potential for transmitter release was near the apparent reversal potential for ICa. 3. When the end EPSP or end IPSP amplitude was plotted against the peak ICa elicited during a presynaptic pulse (peak ICa), large and small depolarizations which evoked the same peak ICa evoked different amounts of transmitter release. The differences in transmitter release were eliminated when end EPSP amplitude was plotted against [symbol: see text] ICa, suggesting that transmitter release during a depolarization depends only upon calcium current and not upon a subsequent voltage‐dependent step. 4. The synaptic transfer function of various measurements of EPSP or IPSP amplitude vs. [symbol: see text]ICa evoked during a presynaptic depolarization was a power function having an exponent of about 3. Similar measurements of EPSP amplitude vs. [symbol: see text]tail ICa evoked following a presynaptic depolarization had an exponent of about 2. 5. Facilitation of an EPSP or IPSP was not due to increases in calcium current at the test depolarization. 6. When the conditioning depolarization was increased and the test depolarization remained constant, EPSP amplitude at the test depolarization and facilitation increased . When the conditioning depolarization remained constant and the test depolarization was increased, EPSP amplitude at the test depolarization increased, while facilitation decreased. 7. Our data suggested that transmitter release at crayfish neuromuscular junctions is a non‐linear function of calcium influx, and that facilitated release utilizes intracellular calcium differently from non‐facilitated release. These data contradict simple models of facilitation which combine the residual calcium hypothesis with the calcium co‐operativity hypothesis of non‐facilitated release.
Oscillatory bursting of phasically firing rat supraoptic neurones in low‐Ca2+ medium: Na+ influx, cytosolic Ca2+ and gap junctions.
Authors: Li Z , Hatton G I
Published: December 2014

1. Whole‐cell patch recordings were obtained from supraoptic nucleus (SON) neurones in horizontal brain slices of adult male rats. Low‐Ca2+ or Ca(2+)‐free perifusion medium induced oscillatory bursting activity in all sixty‐nine cells displaying both phasic firing and depolarizing after‐potentials (DAPs). In fifteen non‐phasic cells without DAPs, Ca(2+)‐free medium produced little or no oscillatory bursting. 2. Typical bursts started with rapid membrane depolarization, resulting in a plateau with superimposed action potentials, and ended several hundred milliseconds later in swift repolarization. Prominent bursting was observed at membrane potentials from ‐50 to ‐70 mV, with maximum amplitudes of 12.2 +/‐ 0.7 mV (mean +/‐ S.E.M.) around ‐70 mV. Development of oscillatory bursting was dependent on reduction of [Ca2+]o, with a threshold for the bursting < or = 1.2 mM Ca2+. 3. Bursting was abolished by addition of TTX, Co2+, Ni2+ or Mg2+ into the Ca(2+)‐free medium, or by replacement of external Na+ with choline or Li+. Low concentrations of TEA or increased [K+]o prolonged burst durations and enlarged oscillation amplitudes. 4. Voltage‐clamp techniques were used to examine the persistent Na+ current (INaP), and revealed that low [Ca2+]o shifted the threshold for INaP activation in a negative direction and enhanced the amplitude of this current. These changes in INaP were abolished by adding Co2+ or Mg2+ to Ca(2+)‐free medium. 5. Direct diffusion of BAPTA or heparin into neurones or bath application of ryanodine suppressed bursting. Oscillations were also eliminated by the uncoupling agents heptanol, halothane or acidification. 6. CNQX, APV, bicuculline, CGP35348 (GABAB receptor antagonist), promethazine, atropine, d‐tubocurarine and suramin had no obvious effects on oscillatory bursting. Blockers of transient Ca2+, or hyperpolarization‐activating cation currents also did not alter bursting activity. 7. These results suggest that intrinsic burst activity in SON neurons perifused with low‐Ca2+ or Ca(2+)‐free medium involves enhanced Na+ influx through persistent Na+ channels, and requires the presence of rapid intracellular Ca2+ mobilization that might also explain the selective existence of oscillatory bursting in phasically firing cells. Intercellular communication through gap junctions appears to be important in determining neuronal activity of the neuroendocrine cells in low‐Ca2+ medium.
Temporal specificity of muscarinic synaptic modulation of the Ca(2+)‐dependent K+ current (ISAHP) in rat hippocampal neurones.
Authors: Zhang L , Han D , Carlen P L
Published: December 2014

1. We examined synaptic modulation of the Ca(2+)‐dependent K+ current (ISAHP), which underlies the slow after‐hyperpolarization (sAHP) in hippocampal CA1 neurones of rat brain slices. ISAHP was evoked in whole‐cell voltage‐clamp mode by depolarizing pulses, and synaptic afferents to CA1 neurones were stimulated electrically with a paired‐pulse protocol. 2. Afferent stimulation delivered 200‐1500 ms prior to be depolarizing pulse produced a profound reduction of ISAHP by 58%, but not other Ca(2+)‐dependent outward currents that preceded ISAHP. Perfusion of slices with atropine significantly attenuated the synaptic reduction of ISAHP, indicating an event mediated largely by muscarinic receptor activation. When delivered < 400 ms after the depolarizing pulse, similar synaptic stimuli produced no substantial reduction in ISAHP, even in neurons where the duration of ISAHP was prolonged to 8‐10 s either by lowering the recording temperature or by intracellular application of a calcium chelator. 3. To examine the effect of cholinergic stimulation of the depolarization‐activated Ca2+ influx, high‐threshold voltage‐activated Ca2+ currents were recorded in the conventional or perforated whole‐cell mode. Perfusion of slices with 5‐10 microM carbachol for 5‐10 min caused no substantial decrease in these Ca2+ currents, suggesting that the synaptic reduction of ISAHP is unlikely to be due to a blockade of depolarization‐induced Ca2+ influx which triggers the generation of ISAHP. 4. The present data demonstrate that afferent stimulation reduces ISAHP only if it occurs prior to the depolarization‐induced Ca2+ influx. We propose that modulation of inactive sAHP channels by muscarinic stimulation may decrease their sensitivity to the influx of Ca2+, whereas sAHP channels activated by Ca2+ may compete with the receptor‐coupled modulation thus rendering the sAHP channels unresponsive to cholinergic afferent stimulation.
Regulation of ram scrotal temperature during heat exposure, cold exposure, fever and exercise.
Authors: Maloney S K , Mitchell D
Published: December 2014

1. We measured body core and scrotal temperatures (Tbody and Tscrotum, respectively) of rams during 5 h of hot (40 degrees C) and cold (6 degrees C) exposure, for 6 h following intravenous injections of saline (0.9% NaCl) or 0.4 micrograms kg‐1 of the purified lipopolysaccharide endotoxin of Salmonella typhosa (LPS), during 40 min of treadmill exercise, and for several days in their pens. 2. At 20‐23 degrees C ambient temperature there were significant, but out of phase, circadian variations in Tbody and Tscrotum. Tscrotum was 3.30 +/‐ 0.03 degrees C lower than Tbody on average. 3. During cold exposure the tunica dartos muscles contracted, nevertheless Tscrotum fell and Tbody‐Tscrotum increased. During heat exposure the tunica dartos muscle relaxed and scrotal sweat glands were activated, nevertheless Tscrotum rose and Tbody‐Tscrotum decreased. There was no change in Tscrotum after LPS injection or during exercise, but Tbody increased in both cases. 4. We suggested that Tscrotum is regulated independently of Tbody via a feedback circuit involving scrotal thermoreceptors and effectors in the form of tunica dartos muscle activity and scrotal sweat gland activity. This local circuit is not affected by adjustments to the general thermo‐regulatory control system during fever. The effector mechanisms were insufficient to maintain Tscrotum during the extremes of heat and cold exposure.
Substance P suppresses GABAA receptor function via protein kinase C in primary sensory neurones of bullfrogs.
Authors: Yamada K , Akasu T
Published: December 2014

1. The effects of substance P (SP) and related tachykinins on the function of gamma‐aminobutyric acid‐A (GABAA) receptors were examined in acutely dissociated neurones of bullfrog dorsal root ganglia (DRG) by using whole‐cell voltage‐clamp techniques. 2. Application of SP (10 nM to 1 microM) depressed inward currents produced by GABAA receptor activation (IGABA). Neurokinin A (NKA) and neurokinin B (NKB) also depressed IGABA; the rank order of agonist potency was SP > NKA > NKB. Spantide ([D‐Arg1, D‐Trp7,9,Leu11]SP) and L‐703,606, NK1 receptor antagonists, blocked the SP‐induced depression of IGABA. 3. SP irreversibly depressed IGABA, when neurones were intracellularly dialysed with GTP gamma S. Intracellular application of GDP beta S prevented the SP‐induced depression of IGABA. Pertussis toxin (PTX) did not block the inhibitory effect of SP on IGABA. 4. The depression of IGABA produced by SP was inhibited by H‐7 and PKC(19‐36), protein kinase C (PKC) inhibitors, but not by H‐9 and HA‐1004, protein kinase A inhibitors. IGABA was suppressed by application of sn‐1,2‐dioctanoyl glycerol (DOG), a PKC activator. 5. It is concluded that activation of neurokinin‐1 (NK1) receptors downregulates the function of the GABAA receptor of primary sensory neurones through a PTX‐insensitive G‐protein. PKC may be involved in the transduction pathway of the tachykinin‐induced inhibition of the GABAA receptor.
Protein kinase C‐mediated enhancement of glycine response in rat sacral dorsal commissural neurones by serotonin.
Authors: Xu T L , Nabekura J , Akaike N
Published: December 2014

1. The modulatory effect of serotonin (5‐hydroxytryptamine, 5‐HT), on the glycine (Gly) response was investigated in neurones acutely dissociated from the rat sacral dorsal commissural nucleus (SDCN) using a nystatin‐perforated patch recording configuration. 2. 5‐HT potentiated the 10(‐5) M Gly‐induced Cl‐ current (IGly) in a concentration‐dependent manner without changing the reversal potential of the Gly response or the affinity of Gly to its receptor. 3. alpha‐Methyl‐5‐HT mimicked and ketanserine blocked the 5‐HT action on IGly, thus indicating the 5‐HT2 receptor‐mediated enhancement. 4. Phorbol‐12‐myristate‐13‐acetate and 1‐oleoyl‐2‐acetylglycerol potentiated IGly. The subsequent application of 5‐HT slightly increase IGly. Chelerythrine blocked the enhancement of IGly by 5‐HT, thus suggesting the involvement of protein kinase C (PKC) in the pathway of 5‐HT action on IGly. 5. Pertussis toxin (IAP) treatment did not block the facilitatory effect of 5‐HT on IGly. 6. BAPTA AM did not disturb the 5‐HT‐induced potentiation of IGly, thus suggesting that [Ca2+]i is not involved in the 5‐HT effect. 7. In conclusion, activation of a 5‐HT2 receptor coupled to an IAP‐insensitive G‐protein increases intracellular diacylglycerol (DAG) formation. The accumulation of DAG also increases the Ca(2+)‐independent PKC activity, thus resulting in the potentiation of the Gly response in the SDCN neurones.
High endogenous calcium buffering in Purkinje cells from rat cerebellar slices.
Authors: Fierro L , Llano I
Published: December 2014

1. The ability of Purkinje cells to rapidly buffer depolarization‐evoked intracellular calcium changes (delta [Ca2+]i) was estimated by titrating the endogenous buffer against incremental concentrations of the Ca(2+)‐sensitive dye fura‐2. 2. In cells from 15‐day‐old rats, pulse‐evoked delta [Ca2+]i were stable during the loading with 0.5 mM fura‐2 through the patch pipette. In cells from 6‐day‐old rats, delta [Ca2+]i decreased by approximately 50% during equivalent experiments. This decrease was not related to changes in Ca2+ influx, since the integral of the Ca2+ currents remained constant throughout the recording. 3. Experiments with high fura‐2 concentrations (1.75‐3.5 mM) were performed in order to obtain for each cell the curve relating delta [Ca2+]i to fura‐2 concentration. From this relationship, values for the Ca2+ binding ratio (the ratio of buffer‐bound Ca2+ changes over free Ca2+ changes) were calculated. 4. In Purkinje cells from 15‐day‐old rats, the Ca2+ binding ratio was approximately 2000, an order of magnitude larger than that of other neurones and neuroendocrine cells studied to date. This Ca2+ binding ratio was significantly smaller (approximately 900) in Purkinje cells from 6‐day‐old rats. 5. We propose that the large Ca2+ binding ratio of Purkinje cells is related to the presence of large concentrations of Ca2+ binding proteins and that these cells regulate their ability to handle Ca2+ loads during development through changes in the concentration of Ca2+ binding proteins.
Characterization of apamin‐sensitive Ca(2+)‐activated potassium channels in human leukaemic T lymphocytes.
Authors: Hanselmann C , Grissmer S
Published: December 2014

1. The whole‐cell recording mode of the patch‐clamp technique was used to study the effect of extracellularly applied ions, toxins and drugs on voltage‐independent, apamin‐sensitive Ca(2+)‐activated K+ channels, K(Ca), expressed in the Jurkat human leukaemic T cell line. 2. Extracellular Ba2+ and Sr+ produced a voltage‐dependent block. The equilibrium dissociation constant of the Ba2+/K(Ca) channel complex increased e‐fold for a 20 mV change of potential. Ba2+ block of Jurkat K(Ca) channels is therefore as steep as expected from the movement of a single divalent cation about half‐way into the electric field of the membrane from the outside. 3. We determined the ion selectivity as well as the conductance of these channels. Calculated permeability ratios, PX/PK, for these K(Ca) channels were 1.0, 0.96, 0.26 and 0.53 for K+, Rb+, Cs+ and NH4+, respectively. Conductance ratios, gX/gK, for the same ions were 1.0, 1.0, 0.67 and 0.11, respectively. Most strikingly this channel can also carry significant current with Cs+ as current carrier. 4. Scyllatoxin (ScTX), a thirty‐one amino acid peptide toxin, reduced current through these K(Ca) channels with a half‐blocking concentration of approximately 0.3 nM independent of the pH. Other drugs that were able to reduce current through these channels include the classical calcium antagonists diltiazem and verapamil. In contrast, nifedipine, clotrimazole and kaliotoxin (100 nM) were unable to block current through these channels in Jurkat T cells.
Acidosis of rat dorsal vagal neurons in situ during spontaneous and evoked activity.
Authors: Trapp S , Lückermann M , Brooks P A , Ballanyi K
Published: December 2014

1. Rat brainstem slices were taken for simultaneous measurements of intracellular pH (pHi) and membrane currents or potentials in dorsal vagal neurons, dialysed with the pH‐sensitive dye BCECF. 2. Intrinsic intracellular buffering power was 18 mM per pH unit, as determined by exposure to trimethylamine in CO2/HCO3(‐)‐free, Hepes‐buffered saline. 3. Tonic spike activity led to a stable fall in pHi of 0.05‐0.2 pH units from a baseline of 7.19 in current‐clamp mode, whereas depolarization from ‐60 to 0 mV for 1 min in voltage‐clamp mode produced an intracellular acidification of 0.3 pH units. The depolarization‐evoked fall in pHi was suppressed by 1 mM Ni2+ or 0.2 mM Cd2+, but not by 0.5 microM TTX or CO2/HCO3(‐)‐free saline. 4. Kainate (100 microM) led to an an inward current of ‐620 pA and a threefold increase in membrane conductance, accompanied by a fall in pHi of 0.33 pH units. 5. GABA (1 mM) evoked a bicuculline‐blockable conductance increase and fall in pHi of up to 0.5 pH units. The GABA‐induced pHi decrease, but not the conductance increase, was suppressed in Hepes solution. 6. Neither tonic spike activity, nor resting current or conductance were markedly changed upon Hepes‐induced intracellular alkalinizations of up to 0.35 pH units, or by an anoxia‐induced fall in pHi of a maximum of 0.36 pH units. 7. The data show that neuronal activity produces profound changes in pHi. It appears that spontaneous spike discharge of dorsal vagal neurons is rather tolerant of major perturbations in pHi.