bluuebunny/crossref_metadata_2025 huggingface dataset:
Region‐specific regulation of neuropeptide mRNAs in rat limbic forebrain neurones by aldosterone and corticosterone.
Authors: Watts A G , Sanchez-Watts G
Published: December 2014

1. We have determined in adrenalectomized male rats the effects of clamping plasma corticosterone and aldosterone at various concentrations on corticotropin‐releasing hormone (CRH), neurotensin/neuromedin N (NT/N) and proenkephalin (pENK) mRNAs in the hypothalamus and amygdala using semi‐quantitative in situ hybridization. 2. Corticosterone differentially regulated the levels of CRH and NT/N but not pENK mRNA. These effects were cell specific. CRH mRNA was reduced in the hypothalamic paraventricular nucleus (PVH), but increased in the central nucleus of the amygdala and bed nuclei of the stria terminalis. NT/N mRNA was never seen in the PVH, whereas levels increased in the central nucleus of the amygdala, but were unaffected in the lateral hypothalamic area. In those regions expressing pENK mRNA, levels were unaffected in all treatment groups. 3. CRH mRNA in both the central nucleus of the amygdala and PVH, and NT/N mRNA in the central nucleus of the amygdala were most sensitive to plasma corticosterone concentrations of less than 120 ng ml‐1, i.e. those seen away from the peak of the diurnal rhythm. In adrenalectomized animals CRH mRNA in both the central nucleus of the amygdala and PVH could be set at levels usually seen in intact animals by the same plasma concentration of corticosterone. 4. The levels of CRH mRNA in the PVH and the central nucleus of the amygdala were closely correlated, while CRH and NT/N mRNA levels were similarly correlated in the central nucleus of the amygdala suggesting the existence of a common regulatory mechanism. The ED50 of their responses to corticosterone and correlations with thymus weight suggested the operation of glucocorticoid (type II) receptor mechanisms. 5. In the absence of corticosterone, aldosterone increased CRH and NT/N mRNA accumulation in the central nucleus of the amygdala, and increased CRH but not NT/N mRNA accumulation in the PVH. Aldosterone also blunted the dose‐response effects of corticosterone on CRH and NT/N mRNA levels in the central nucleus of the amygdala, but not in the PVH. 6. These results suggest that, in intact animals, adrenal steroids play a major role in maintaining the levels of neuropeptide mRNAs in the PVH, bed nuclei of the stria terminalis and central nucleus of the amygdala. The results underscore the importance of cell‐specific mechanisms operating to regulate the expression of neuropeptide genes in different cell types in response to diverse physiological conditions.
Thrombin‐induced inhibition of potassium currents in human retinal glial (Müller) cells.
Authors: Puro D G , Stuenkel E L
Published: December 2014

1. Glial cells are known to play a role in regulating the microenvironment of the nervous system. While earlier considerations of glial function assumed a passive, static physiology for these cells, this is not likely to be the case. In this study, we begin to examine how the physiology of Müller glial cells changes in response to molecules in the microenvironment. 2. Perforated‐path recordings and intracellular calcium measurements were performed on human retinal Müller cells in vitro. 3. Analysis of whole‐cell currents revealed that the human Müller glial cells have an inwardly rectifying K+ current (IK(IR) which is active near the resting membrane potential. This IK(IR) is significantly inhibited when the Müller cell is exposed to thrombin, a molecule that is likely to enter the retina with a breakdown of the blood‐retinal barrier and may be endogenous to the nervous system. 4. A variety of experiments point to a role for Ca2+ as a second messenger mediating the inhibitory effect of thrombin on the IK(IR) of Müller cells. Specifically, thrombin evokes an increase in intracellular [Ca2+] in the Müller cells; the Ca2+ chelator BAPTA blocks the effects of thrombin on both the inhibition of IK(IR) and the rise in intracellular [Ca2+]; exposure to ionomycin, a calcium ionophore, induces a reduction in the IK(IR) of Müller cells. 5. A thrombin‐ induced inhibition in the IK(IR) of Müller cells is likely to have significant functional consequences for the retina since these ion channels are involved in K+ homeostasis. 6. Our experiments support the idea that the physiology of Müller glial cells is dynamic and can be markedly affected by molecules in the microenvironment.
Hypoxic changes in rat locus coeruleus neurons in vitro.
Authors: Nieber K , Sevcik J , Illes P
Published: December 2014

1. Intracellular recordings were made in a pontine slice preparation of the rat brain containing the nucleus locus coeruleus (LC). Locus coeruleus neurons responded to brief hypoxic stimuli (replacement of 95% O2‐5% CO2 with 95% N2‐5% CO2) with hyperpolarization and a cessation of spontaneous action potentials. When the cells were continuously hyperpolarized by about 15 mV in order to abolish spontaneous firing, hypoxia induced an early depolarization (HD), followed by a hypoxic hyperpolarization (HH) and after reoxygenation, a posthypoxic hyperpolarization (PHH). These responses were accompanied by a decrease in input resistance, which was larger during HH than during HD but, thereafter, became smaller during PHH. 2. The hypoxia‐induced currents associated with the changes in membrane potential, at a holding potential of ‐70 mV, were an early inward current (HIC), a subsequent outward current (HOC) and after reoxygenation, another outward current (PHOC). The HIC did not change with an increasing holding potential. In contrast, the HOC reversed its amplitude at about ‐95 mV. Finally, the PHOC decreased, but did not reverse its polarity at more negative holding potentials. When the external K+ was elevated from 2.5 to 10.5 mM, the current‐voltage (I‐V) relation of the HOC and its reversal potential were shifted to the right. 3. In the presence of tetrodotoxin, the HH decreased. A low Ca(2+)‐high Mg2+ medium depressed both the HH and PHH. Rauwolscine did not alter either response to hypoxia, while 8‐cyclopentyl‐1,3‐dipropylxanthine decreased the PHH only. S‐(p‐Nitrobenzyl)‐6‐thioguanosine potentiated both HH and PHH. 4. Whereas tolbutamide markedly lowered the HH and PHH, glibenclamide was ineffective. Tetraethylammonium also failed to alter the hypoxic responses. Furthermore, ouabain or the removal of K+ from the superfusion medium, depressed PHH. 5. Pressure application of adenosine inhibited the spontaneous firing of LC neurons. DPCPX did not alter the firing, but antagonized the effect of adenosine. Tolbutamide also counteracted the inhibitory effect of adenosine and, additionally, facilitated the firing rate in some neurons. Moreover, tolbutamide abolished the adenosine‐induced outward current. 6. Early hypoxic depolarization and PHH are mostly due to the blockade and subsequent reactivation of the K(+)‐Na+ pump, respectively. The HH is caused by the opening of ATP‐sensitive K+ (KATP) channels in response to the hypoxia‐induced decline of intracellular ATP. Adenosine released by hypoxic stimuli may lead to an adenosine A1‐receptor‐mediated opening of (KATP) channels during the HH and more markedly during the PHH.
Influence of permeating ions on potassium channel block by external tetraethylammonium.
Authors: Ikeda S R , Korn S J
Published: December 2014

1. Block by external tetraethylammonium (TEA) was examined on currents carried by potassium (K+) and sodium (Na+) through the cloned delayed rectifier K+ channel Kv2.1. 2. In the presence of high intracellular K+ and high extracellular Na+, currents through the Kv2.1 channel were carried almost exclusively by K+. In the absence of both intracellular and extracellular K+, large currents were carried by Na+ through the Kv2.1 channel. 3. External application of 30 mM TEA blocked K+ currents through Kv2.1 by 87%. The same external concentration of TEA had no effect on Na+ currents through this channel. 4. As the K+ concentration was increased between 0 and 140 mM, the percentage of current blocked by TEA progressively increased from 0 to 87%. 5. These data indicate that block of K+ channels by external TEA depends on the permeating ion. Furthermore, these data indicate that in the presence of Na+ and absence of K+, TEA does not bind to the channel, and that addition of low concentrations of K+ facilitates TEA binding.
Different types of ganglion cell in the cardiac plexus of guinea‐pigs.
Authors: Edwards F R , Hirst G D , Klemm M F , Steele P A
Published: December 2014

1. Intracellular recordings were made from the parasympathetic ganglion cells that lie in the epicardium of the left atrium of guinea‐pig heart near the interatrial septum. 2. Three distinct types of neurone were identified on the basis of their electrophysiological properties. In one group of neurones, S cells, somatic action potentials were followed by brief after‐hyperpolarizations. In the other two sets of neurones, somatic action potentials were followed by prolonged after‐hyperpolarizations. The neurones with prominent after‐hyperpolarization were further subdivided: one group of neurones, P cells, showed inward rectification at membrane potentials near the resting membrane potential whilst neurones in the other group, SAH cells, did so only at more negative potentials. 3. In the group of neurones that displayed inward rectification at potentials near rest, rectification resulted from the activation of an inward current, which resembled the hyperpolarization‐activated inward current present in cardiac muscle pacemaker cells. 4. The three different types of neurone received different patterns of synaptic input. Each SAH cell received a synaptic excitatory connection from the vagus which in most cells released sufficient transmitter to initiate an action potential in that cell; several SAH cells also received a separate connection, which could be activated by local stimulation. Although most S cells failed to receive a synaptic input from the vagus, all of those tested received an excitatory synaptic input which could be activated by local stimulation. Virtually all P cells failed to receive a synaptic input from the vagus; in addition, local stimulation failed to initiate synaptic potentials in P cells. 5. When the structure of cardiac ganglion cells was determined, by loading the cells with either biocytin or neurobiotin, it was found that most cells lacked extensive dendritic processes. S cells were invariably monopolar, most P cells were dipolar or pseudodipolar, whereas many SAH cells were multipolar. 6. In many neurones an on‐going discharge of action potentials was detected in the absence of obvious stimulation. In S and SAH cells, the action potentials resulted from an on‐going discharge of excitatory synaptic potentials. However, when a spontaneous discharge of action potentials was detected in P cells a discharge of excitatory synaptic potentials was not detected. 7. The results are discussed in relation to the idea that the three different types of cell may have different functions and that some of the cells may be organized in such a way as to permit the local handling of neuronal information within the heart.
Disynaptic group I excitation of synergist ankle extensor motoneurones during fictive locomotion in the cat.
Authors: McCrea D A , Shefchyk S J , Stephens M J , Pearson K G
Published: December 2014

1. Intracellular recording from medial gastrocnemius (MG) motoneurones was used to examine postsynaptic potentials produced by electrical stimulation of the plantaris nerve at group I strength at rest and during fictive locomotion. Fictive locomotion was evoked by stimulation of the midbrain locomotor region (MLR) in decerebrate cats or in decerebrate, acute low‐spinal cats by perineal stimulation following intravenous administration of clonidine and naloxone. 2. In both MLR and spinal fictive locomotor preparations, stimulation of plantaris nerve group I afferents at rest evoked short‐latency (< 2 ms) IPSPs in MG motoneurones. During the extensor phase of MLR‐evoked locomotion, the same stimulation produced short‐latency (1.6‐1.8 ms) EPSPs. Such latencies suggest mediation by one interneurone interposed between plantaris nerve group I afferents and MG motoneurones. Non‐monosynaptic, short‐latency excitation was not seen at rest nor during the flexion phase of the step cycle. 3. Group I EPSPs during the extensor phase of MLR‐evoked locomotion were evoked by stimulation at intensities ranging from 1.4‐2 times threshold (T). The effectiveness of stimulation intensities < 1.5 T suggests that activation of group II afferents is not required to evoke disynaptic excitation. Selective activation of group Ia afferents by stretches of the Achilles tendon also produced disynaptic EPSPs during extension. 4. During fictive locomotion in spinal animals pretreated with clonidine, short‐latency group I EPSPs were not seen but group I IPSPs recorded at rest disappeared or were greatly attenuated. The failure of depolarizing current to reveal group I IPSPs suggests that fictive locomotion involves an inhibition of the inhibitory interneurones that operate at rest. In both clonidine‐treated spinal and MLR preparations, trains of stimuli at group I strength evoked longer‐latency and slowly rising potentials that were more prominent during the flexor phase of fictive locomotion. 5. These results show a reduction in short‐latency group I inhibition of synergists in both MLR and clonidine‐treated spinal preparations during fictive locomotion. In addition, activation of group I afferents evokes short‐latency excitation of synergists during extension in the MLR preparation. Such excitatory reflexes activated by ankle extensor group Ia and Ib afferents may form an excitatory feedback system, reinforcing on‐going extensor activity during the stance phase of the step cycle.
Local Ca2+ transients (Ca2+ sparks) originate at transverse tubules in rat heart cells.
Authors: Shacklock P S , Wier W G , Balke C W
Published: December 2014

1. The origins of local [Ca2+]i transients (Ca2+ sparks) were studied using dual‐channel confocal laser scanning microscopy. Line scan images showing [Ca2+]i (as fluo‐3 fluorescence) and the transverse tubule membranes (as Di‐8 fluorescence) were obtained simultaneously in single rat cardiac ventricular cells. 2. Line scan images of Di‐8 fluorescence showed peaks regularly spaced at intervals of 1.83 +/‐ 0.30 microns (mean +/‐ S.D.). These peaks corresponded to the transverse tubules (T‐tubules) in cross‐section. 3. Line scan images of fluo‐3 fluorescence showed local [Ca2+]i transients (LCTs or Ca2+ sparks) evoked by electrical stimulation. 4. Eighty‐five per cent (85%) of all Ca2+ sparks evoked by electrical stimulation (n = 138, in 5 cells) occurred within 0.5 micron of a T‐tubule. Thirty per cent (30%) occurred within 1 pixel (0.20 micron) of a T‐tubule. 5. In some cells studied (3 out of 5), certain T‐tubules had a higher probability of being sites of origin of Ca2+ sparks than others. 6. These results support local control theories of excitation‐contraction coupling in which Ca2+ release from the sarcoplasmic reticulum (SR) is triggered by a high local [Ca2+]i established between the L‐type Ca2+ channels in the T‐tubules and associated ryanodine receptor(s) in the junctional SR.
GABAergic and glutamatergic effects on behaviour in fetal sheep.
Authors: Bissonnette J M , Hohimer A R , Knopp S J
Published: December 2014

1. Studies were carried out in unanaesthetized fetal sheep at 125‐135 days gestation to investigate neurotransmitters involved in behavioural state. 2. Catheters and electrodes were chronically placed to record tracheal and arterial pressure, electrocortical activity (ECoG), nuchal muscle activity and to instill drugs into the cerebrospinal fluid (CSF) of the fourth ventricle. 3. Administration of the N‐methyl‐D‐aspartate receptor antagonists DL‐2‐amino‐5‐phosphopentanoic acid (AP5) or (+)‐5‐methyl‐10,11‐dihydro‐5H‐dibenzol[a,d]cyclohepten‐5,10‐ iminemoleate (MK‐801) increased the incidence of fetal behaviour characterized by low voltage ECoG, nuchal muscle activity and an increase in mean arterial blood pressure from 4.1 +/‐ 6 to 60.6 +/‐ 6.2% (mean +/‐ S.E.M.) (AP5; P = 0.003) and from 7.6 +/‐ 3.6 to 50.8 +/‐ 7.0% (MK‐801; P = 0.004; values are expressed as the percentage of each 60 min period in which the state was present). 4. The incidence of fetal breathing during periods of low voltage (LV)‐ECoG and nuchal muscle activity was 83.1 +/‐ 5.6%. The incidence of fetal breathing during LV‐ECoG associated with nuchal muscle atonia was 63.1 +/‐ 5.0% before AP5 or MK‐801 and 64.4 +/‐ 9.8% after instillation of these drugs. The amplitude of fetal breaths increased from 4.0 +/‐ 0.3 mmHg in low voltage ECoG periods to 6.7 +/‐ 0.8 mmHg (P = 0.006) during periods of low voltage with nuchal muscle activity. There was no significant change in breath timing during these periods.(ABSTRACT TRUNCATED AT 250 WORDS)
Time course of ‘set’‐related changes in muscle responses to stance perturbation in humans.
Authors: Schieppati M , Nardone A
Published: December 2014

1. In standing subjects, toe‐down rotation of a supporting platform elicits a medium‐latency response (MLR) in tibialis anterior (TA) muscle and a long‐latency response (LLR) in soleus (Sol). Toe‐up rotation induces a short‐latency response (SLR) in Sol and a LLR in TA. When subjects steadily hold onto a stable frame, all responses are decreased, except Sol SLR. The aim of this investigation was to assess whether the response modulation is dependent on information from the hand touching the frame, or whether it anticipates the holding task. 2. The time course of the changes in response amplitude was studied in a time interval centred around the act of holding, performed in a reaction‐time mode. Subjects kept their extended arm close to the frame in front of them and brought the hand in contact with the frame in response to a visual go‐signal. The platform was moved at different intervals prior to or after the go‐signal. Surface EMGs of Sol, TA and deltoid (Delt) were recorded. 3. TA MLR began to decrease when the platform was displaced at an interval of 140 ms after the go‐signal, about 200 ms before subjects touched the frame and 120 ms before termination of Delt EMG. Four hundred milliseconds after the go‐signal the response reached and maintained maximal inhibition, similar to that occurring under the stationary holding condition. The time course of inhibition of Sol LLR and TA LLR was similar to that of TA MLR, except that LLRs began to decrease at an earlier interval. Due to the different response latency from the onset of the perturbations, the beginning of inhibition of both MLRs and LLRs occurred almost simultaneously. 4. The changes in amplitude of leg muscle responses are not triggered by the go‐signal, contact with the frame, or arm motion, suggesting that the modulation is related to the transition to a new, stabilized postural ‘set’. The similar extent and parallel time course of MLR and LLR suppression, possibly transmitted through different pathways, points to the spinal cord as the site of action. The lack of depression of the monosynaptic SLR suggests an effect at premotoneuronal level. On the basis of selectivity, latency and time course of the effect, we favour the hypothesis that a monoaminergic pathway from the brainstem is involved.
Effect of insulin on Na+,K(+)‐ATPase in rat collecting duct.
Authors: Féraille E , Rousselot M , Rajerison R , Favre H
Published: December 2014

1. The collecting duct is involved in the whole antinatriuretic effect of insulin, as indicated in vitro by the stimulatory effect of the hormone on ouabain‐sensitive 86Rb+ uptake. Since Na+,K(+)‐ATPase drives Na+ reabsorption, the contribution of the Na+ pump to the effect of insulin was investigated in rat isolated cortical and outer medullary collecting duct. 2. Insulin enhanced ouabain‐sensitive 86Rb+ uptake in the absence, as well as in the presence, of either 5 x 10(‐4) M amiloride or 10(‐3) M hydrochlorothiazide (HCT). Maximal ouabain‐sensitive 86Rb+ uptake, measured in Na(+)‐loaded tubules, was also enhanced by insulin. The insulin effect persisted both in the absence of external Na+, when the Na+,K(+)‐ATPase operates in a Rb(+)‐Rb+ exchange mode, and in tubules depolarized by a high external concentration (20 mM) of Rb+ or by addition of 3 mM Ba2+. 3. Insulin treatment did not alter the intracellular Na and K concentrations, the specific binding of [3H]ouabain measured in intact tubules, or the hydrolytic activity of Na+,K(+)‐ATPase measured after permeabilization of the tubule cells. 4. In conclusion, in the rat collecting duct, insulin increased Na+,K(+)‐ATPase‐mediated cation transport independently of Na+ availability, membrane potential and recruitment of pump units. The effect of insulin was lost after cell permeabilization, suggesting the presence of a cytosolic factor which controls the turnover of Na+,K(+)‐ATPase.
Depolarizing GABA‐activated Cl‐ channels in embryonic rat spinal and olfactory bulb cells.
Authors: Serafini R , Valeyev A Y , Barker J L , Poulter M O
Published: December 2014

1. We have compared the electrical properties of the Cl‐ channels activated by GABA in cells acutely dissociated from embryonic (E) spinal cord (SC) and olfactory bulb (OB) regions at E15 using different configurations of the patch‐recording technique. By in situ analysis these cells express GABAA receptor mRNAs encoding a common set of subunits (alpha 2, beta 2, and beta 3). SC cells also express alpha 3, alpha 5 and gamma 2s transcripts. 2. Whole‐cell recordings revealed current responses to GABA (0.5 microM to 1 mM) in 242 out of 294 cells. In both SC and OB cells, currents evoked by 2 microM GABA could be potentiated by diazepam (DZP) in a dose‐dependent manner with an EC50 of approximately 50 nM in both SC and OB. The maximal effect was approximately 300%. Both SC and OB cells exhibited GABA‐activated currents that were only partially sensitive to zinc even at high micromolar concentrations. The effect of DZP and the relatively modest sensitivity to zinc suggest the presence of gamma subunits in both preparations. 3. Spectral analysis of current responses in twenty‐six cells showed that power spectra could be fitted by three exponential components (tau 1‐3) in the cells of both areas. The tau of the longest‐lasting component (tau 3) was significantly different in the cells of the two areas: approximately 50 ms in OB and 80‐100 ms in SC. No statistically significant differences in the average inferred unitary conductance between the two cell types could be resolved. 4. Single‐channel properties were examined directly using the cell‐attached configuration. GABA‐activated channels could be recorded in only 89 out of well‐sealed 984 patches and most of them exhibited multiple channel activity. The mean open time in the response to 10 microM GABA was significantly shorter in OB cells (12 ms) compared to SC cells (25 ms) while the average conductance values were not significantly different between the two cell types. 5. On average, Cl‐ channels reversed polarity when the on‐cell patch pipette potential was approximately ‐30 mV. Thus, in these embryonic neurons, micromolar GABA activates Cl‐ channels, which, when open, effectively depolarize cells by approximately 30 mV. 6. Cl‐ channels activated by GABA are open longer in embryonic SC cells than in OB cells. This statistically significant difference in native GABAA receptor Cl‐ channel properties correlates with, and may be related to differences in subunit mRNA expression.
Synaptic control of rat supraoptic neurones during osmotic stimulation of the organum vasculosum lamina terminalis in vitro.
Authors: Richard D , Bourque C W
Published: December 2014

1. The effects of osmotic or electrical stimulation of the organum vasculosum lamina terminalis (OVLT) were examined during intracellular recordings (32 degrees C) obtained from ninety‐five supraoptic nucleus magnocellular neurosecretory cells (MNCs) in superfused explants of rat hypothalamus. 2. Brief (10‐20 s) applications of hypertonic and hypotonic solutions to the area of the OVLT caused prolonged (> 1 min) increases and decreases, respectively, in electrical activity in seventy of seventy‐four trials performed on neurones with membrane potentials near spike threshold (approximately ‐55 mV). Changes in firing frequency were related to changes in external osmolality in a dose‐dependent manner between 275 and 355 mosmol kg‐1. 3. When 30 s periods recorded immediately before, and 30 s following, the application of an osmotic stimulus were examined, the frequency of spontaneous EPSPs (sEPSPs) was related in a dose‐dependent manner to the osmolality of the solution superfusing the OVLT region. The increased EPSP frequency was maintained and did not adapt if the osmolality of the medium was raised for periods of > 10 min. In contrast, the frequency of spontaneous IPSPs (sIPSPs) was virtually unaffected by changes in external osmotic pressure. 4. Osmotically evoked changes in MNC firing were strongly correlated with accompanying changes in the frequency of sEPSPs (slope, 0.9; correlation coefficient (r) = 0.7), but not sIPSPs (r = 0.2), suggesting that changes in firing rate following osmotic stimulation of the OVLT are selectively mediated by changes in synaptic excitation. 5. In the presence of bicuculline (5‐10 microM), electrical stimulation of the OVLT evoked fast EPSPs in forty‐seven of forty‐eight MNCs tested. These responses were reversibly reduced by application of 20‐40 microM kynurenic acid (n = 3) or 20‐40 microM 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX; n = 11). Similarly, bath application of CNQX (n = 3) or kynurenic acid (n = 4) reversibly abolished the excitatory response of supraoptic neurones following hypertonic stimulation of the OVLT. 6. Brief (10‐15 s) applications of gamma‐aminobutyric acid (GABA) over the OVLT reversibly abolished increases in sEPSP frequency and action potential firing rate evoked by hyperosmotic stimulation of the OVLT. In the presence of GABA, the rates of sEPSP and sIPSP frequency were reduced to 37 +/‐ 10 and 44 +/‐ 13% (means +/‐ S.E.M.), respectively, of those observed under isotonic conditions (295 mosmol kg‐1). 7. These results suggest that inhibitory and excitatory pathways originating from neurones located within the OVLT are tonically active under resting osmotic conditions in rat hypothalamic explants. Osmotically evoked changes in MNC firing, however, are selectively mediated through increases or decreases in the intensity of the excitatory component of OVLT‐derived inputs.
Calcium‐induced calcium release in rat sensory neurons.
Authors: Shmigol A , Verkhratsky A , Isenberg G
Published: December 2014

1. In isolated dorsal root ganglion cells (DRG neurons), changes in the concentration of global cytosolic Ca2+ (delta [Ca2+]c) were measured by the fluorescence of K5‐indo‐1. Depolarizations from ‐60 to 0 mV (500 ms) and Ca2+ influx through Ca2+ channels (ICa) increased [Ca2+]c by 480 +/‐ 113 nM, the peak occurring 542 +/‐ 76 ms (mean +/‐ S.E.M.) after repolarization. 2. Ryanodine (10 microM) reduced depolarization‐induced delta [Ca2+]c by up to 80% and blocked delta [Ca2+]c induced by 20 mM caffeine. 3. Peak delta [Ca2+]c and peak ICa followed a similar bell‐shaped voltage dependence. Removal of extracellular Ca2+ abolished depolarization‐induced delta [Ca2+]c; its elevation from 2 to 8 mM increased peak ICa by 30% and delta [Ca2+]c by 108%. 4. Ca2+ influx at 0 mV was graded by pulse durations between 20 and 500 ms. Up to 200 ms, delta [Ca2+]c increased linearly with Ca2+ influx. Depolarizations longer than 200 ms induced a supralinear increase in delta [Ca2+]c that was abolished by caffeine (20 mM). 5. The supralinear increase in delta [Ca2+]c and the caffeine‐induced delta [Ca2+]c were measured only in thirteen of nineteen DRG neurons; in the other six of nineteen cells both properties were absent. The results suggest that Ca(2+)‐induced Ca2+ release (CICR) is expressed differently in different populations of DRG neurons. 6. A single action potential did not significantly increase [Ca2+]c. Trains of stimuli (20 Hz) induced delta [Ca2+]c that linearly increased with the number of action potentials. Delta [Ca2+]c due to 100 action potentials had a significant ryanodine‐sensitive component. 7. It is discussed that CICR can contribute to the depolarization‐induced [Ca2+]c, provided the Ca2+ influx lasts for a certain minimum period of time.
Kinetic evidence distinguishing volume‐sensitive chloride current from other types in guinea‐pig ventricular myocytes.
Authors: Shuba L M , Ogura T , McDonald T F
Published: December 2014

1. Kinase‐mediated chloride currents (ICl) in guinea‐pig ventricular myocytes were activated by application of phorbol ester or forskolin, and compared with currents induced by hyposmotic swelling. Swelling‐activated current was identified as ICl from changes in reversal potential, outward rectification and conductance when the Cl‐gradient was modified. 2. Kinase‐stimulated currents were relatively time and voltage independent, whereas hyposmotic swelling‐stimulated (hyposmotic‐stimulated) currents inactivated during 100 ms pulses to positive potentials. Forskolin stimulated time‐independent ICl in myocytes with current unresponsive to hyposmotic superfusion, and superimposed a similar pedestal on time‐dependent ICl in swollen myocytes. 3. Less negative holding potentials depressed hyposmotic‐stimulated ICl tested at +80 mV; inhibition was half‐maximal at ‐25 mV. Pulses from ‐80 to +80 mV inactivated up to 75% of ICl along a multi‐exponential time course; repolarization elicited inwardly developing tail currents whose time courses suggest complex gating. 4. Hyperpolarizations, after strongly‐inactivating depolarizations, triggered reactivating tail currents whose amplitude and configuration were dependent on voltage and Cl‐gradients; tails were large and inwardly developing at potentials negative to the calculated Cl‐equilibrium potential (ECl), small and outwardly developing at potentials positive to ECl, and time independent near ECl. 5. These results suggest that the volume‐sensitive Cl‐ channels investigated here are distinct from other Cl‐ channels in guinea‐pig ventricular myocytes. However, their voltage‐dependent properties strongly resemble those of volume‐sensitive Cl‐ channels in certain epithelial cells.
Effect of CO2 on the metabolic and ventilatory responses to ambient temperature in conscious adult and newborn rats.
Authors: Saiki C , Mortola J P
Published: December 2014

1. In newborn and adult rats, hypoxia decreases metabolic rate, especially at low ambient temperatures (Ta). We examined whether a similar effect can occur during hypercapnia. 2. We measured metabolism (oxygen consumption, Vo2, open flow‐through method), and expiratory ventilation (VE; barometric method (adults), airflow plethysmograph (newborns)) in air and 2% or 5% CO2 in normoxia. 3. In adults, Vo2 was higher at Ta = 10 degrees C than 25 degrees C. At each Ta, CO2 breathing did not change Vo2, but increased VE, less at 10 degrees C (up to +100%) than at 25 degrees C (+161%). Blood pressure was maintained at both values of Ta and CO2, while pulse rate and body temperature were decreased in 5% CO2 at 10 degrees C. 4. In newborns, the metabolic response to lowering Ta (from 40 to 20 degrees C) much depended on behavioural responses, being larger in groups of two or four pups than in individual animals. In no case did CO2 influence the response. VE increased during 5% CO2 exposure, more so at Ta = 33 percent C (+69%) than at 25 degrees C (+49%). 5. In both adults and newborns, hypoxia (10% O2) always decreased metabolic rate. 6. We conclude that hypercapnia has no appreciable effects on metabolic rate in rats (both newborns and adults) even at low Ta, a result quite different from the hypometabolic response to hypoxia.
Activation and blockade of mouse muscle nicotinic channels by antibodies directed against the binding site of the acetylcholine receptor.
Authors: Bufler J , Kahlert S , Tzartos S , Toyka K V , Maelicke A , Franke C
Published: December 2014

1. Using the patch‐clamp technique, we have found that mouse muscle nicotinic acetylcholine receptor (nAChR) channels can be activated by low concentrations of a monoclonal antibody (MoAb), referred to as WF6, which is directed against the acetylcholine (ACh) binding site. Similar effects were seen using IgG or F(ab)2 fragments from the sera of patients with myasthenia gravis (MG), which contain polyclonal anti‐nAChR antibodies. 2. The mean open times of MoAb and the slope conductance of single WF6‐activated single channels were similar to those of ACh‐activated channels under the same experimental conditions. 3. On outside‐out patches, single channel activity was elicited by MoAb WF6 and MG F(ab)2 fragments, and was blocked by (+)‐tubocurarine. We therefore concluded that MoAb WF6 and the MG F(ab)2 fragments activate the nAChR. 4. MoAb WF6 and MG F(ab)2 fragments blocked the current activated by pulsed application of 10(‐4) M ACh to a significant extent. The block was partly reversible. The rate constants for the binding and dissociation of MoAb WF6 from the receptor were determined quantitatively.
Chemosensitive medullary neurones in the brainstem‐‐spinal cord preparation of the neonatal rat.
Authors: Kawai A , Ballantyne D , Mückenhoff K , Scheid P
Published: December 2014

1. Using the isolated medulla and spinal cord of the neonatal rat, the response to CO2‐induced changes in superfusate pH was examined in whole cell and perforated patch recordings from ventral medullary neurones which were identified by injection of Lucifer Yellow. The respiratory response to changing the CO2 concentration (from 2 to 8%) consisted of an increase in phrenic burst frequency, which could be accompanied by an increase, decrease or no change in burst amplitude. 2. Five classes of neurone ‐ inspiratory, post‐inspiratory, expiratory, respiration‐modulated and ionic ‐ were distinguished on the basis of their membrane potential and discharge patterns. Almost all (112 of 123) responded rapidly to 8% CO2 with a sustained change in membrane potential. Depolarizing responses (3‐18 mV) occurred in inspiratory, respiration‐modulated and 45% of tonic neurones. Hyperpolarizing responses (2‐19 mV) occurred in expiratory and post‐inspiratory neurones. The remaining tonic neurones were inhibited or showed no response. 3. In representatives of each class of neurone, membrane potential responses to 8% CO2 were retained when tested in the presence of tetrodotoxin (n = 7), low (0.2 mM) Ca(2+)‐high (5 mM) Mg2+ (n = 23) or Cd2+ (0.2 mM) (n = 3)‐containing superfusate, implying that they are mediated by intrinsic membrane or cellular mechanisms. 4. Neurones were distributed between 1200 microns rostral and 400 microns caudal to obex, and their cell bodies were located between 50 and 700 microns below the ventral surface (n = 104). Almost all responsive neurones (n = 78) showed dendritic projections to within 50 microns of the surface. 6. These experiments indicate that significant numbers of ventral medullary neurones, including respiratory neurones, are intrinsically chemosensitive. The consistency with which these neurones show surface dendritic projections suggests that this sensitivity may arise in part at this level.
Postnatal development of the pattern of respiratory and cardiovascular response to systemic hypoxia in the piglet: the roles of adenosine.
Authors: Elnazir B , Marshall J M , Kumar P
Published: December 2014

1. In 3‐day‐old and 3‐week‐old spontaneously breathing piglets anaesthetized with Saffan, we have studied ventilatory and cardiovascular responses evoked by 5 min periods of hypoxia (breathing 10 and 6% O2). 2. In 3‐day‐old piglets both 10 and 6% O2 evoked an increase followed by a secondary fall in ventilation, a gradual tachycardia and a renal vasoconstriction, with an increase in femoral blood flow that was attributable to femoral vasodilatation. Arterial blood pressure rose initially but fell towards control values by the 5th minute of hypoxia. 3. The stable adenosine analogue 2‐chloroadenosine (2‐CA; 30 mg kg(‐1) i.v.) evoked bradycardia and renal vasoconstriction, but had no effect on femoral vasculature. These responses were blocked by the adenosine receptor antagonist 8‐phenyltheophylline (8‐PT; 8 mg kg(‐1) i.v.). 8‐PT also abolished the secondary fall in ventilation evoked by 10 and 6% O2 and the renal vasoconstriction evoked by 10% O2, but had no effect on the tachycardia, or on the femoral vascular response. 4. By contrast, in 3‐week‐old piglets both 10 and 6% O2 evoked a sustained increase in ventilation, tachycardia and a rise in arterial pressure with renal vasoconstriction, but no change in renal blood flow and substantial femoral vasodilatation with an increase in femoral blood flow. 2‐CA evoked bradycardia and renal vasoconstriction, as in 3‐day‐old piglets, but also evoked pronounced femoral vasodilatation. 8‐PT blocked these responses and the hypoxia‐induced femoral vasodilatation, but had no significant effect on other components of the hypoxia‐induced response. 5. We propose that there is postnatal development of the ventilatory and cardiovascular responses evoked by systemic hypoxia and of the role of locally released adenosine in these responses: at 3 days, adenosine released within the central nervous system and within the kidney is a major contributor to the secondary fall in ventilation and renal vasoconstriction respectively, whereas at 3 weeks, adenosine makes little contribution to the ventilatory response, or renal vasoconstriction, but is largely responsible for hypoxia‐induced vaso‐dilatation in skeletal muscle.
Contribution of a non‐inactivating potassium current to the resting membrane potential of fusion‐competent human myoblasts.
Authors: Bernheim L , Liu J H , Hamann M , Haenggeli C A , Fischer-Lougheed J , Bader C R
Published: December 2014

1. Using the patch‐clamp technique, a new non‐inactivating voltage‐gated potassium current, IK(ni), was studied in cultured fusion‐competent human myoblasts. 2. IK(ni) is activated at voltages above ‐50 mV and its conductance reaches its maximum around +50 mV. Once activated, the current remains at a steady level for minutes. 3. Reversal potential measurements at various extracellular potassium concentrations indicate that potassium ions are the major charge carriers of IK(ni). 4. IK(ni) is insensitive to potassium channel blockers such as charybdotoxin, dendrotoxins, mast cell degranulating (MCD) peptide, 4‐aminopyridine (4‐AP), 3,4‐diaminopyridine (3,4‐DAP) and apamin, but can be blocked by high concentrations of TEA and by Ba2+. 5. A potassium channel of small conductance (8.4 pS at +40 mV) with potential dependence and pharmacological properties corresponding to those of IK(ni) in whole‐cell recording is described. 6. IK(ni) participates in the control of the resting potential of fusion‐competent myoblasts, suggesting that it may play a key role in the process of myoblast fusion.
The incidence of initial doublets in the discharges of motoneurones of two different inspiratory muscles in the cat.
Authors: Kirkwood P A , Munson J B
Published: December 2014

1. Trains of action potentials in motoneurones frequently commence with an initial doublet; i.e. a uniquely short interspike interval. Previous authors have speculated on the functional importance of initial doublets. Here we test the hypotheses that these doublets are associated with particular classes of motoneurones or particular physiological conditions. 2. Discharges of inspiratory motoneurones were recorded extracellularly in the thoracic ventral horn of anaesthetized, paralysed cats. Seventy units (35 each with axons in the internal and external intercostal nerves) were classified on the basis of their maximum firing rates, start times in the respiratory cycle and axonal destination. 3. Initial doublets were defined by an interspike interval < 14 ms. Of seventeen units firing initial doublets, fifteen had axons in the external intercostal nerve and two had axons in the internal intercostal nerve. Neither maximum firing rate nor start time during the respiratory cycle predicted the occurrence of doublets. 4. The chemical drive to breathe was manipulated by altering the CO2 content of the inspired gas or by briefly stopping the respiratory pump. Varying the chemical drive to breathe had no consistent effect on the occurrence of initial doublets. 5. These results support the view that initial doublets are part of the normal pattern of discharge of motoneurones. However, because the incidence of doublets does not consistently support previous functional hypotheses, we argue that the occurrence of doublets may not necessarily be dictated by the CNS, but in some circumstances it is an epiphenomenon dependent on the state of the motoneurone, in particular on the statistical properties of its synaptic inputs.
Cooling inhibits exocytosis in single mouse pancreatic B‐cells by suppression of granule mobilization.
Authors: Renström E , Eliasson L , Bokvist K , Rorsman P
Published: December 2014

1. The mechanisms by which cooling inhibits insulin secretion were investigated by capacitance measurements of exocytosis in single mouse pancreatic B‐cells maintained in short‐term tissue culture. 2. A reduction of the bath temperature from 34 to 24 degrees C produced a gradual inhibition of exocytosis. Inhibition of exocytosis was use dependent rather than time dependent. The steady‐state inhibition amounted to 90%, which was paralleled by a 30% reduction of the peak Ca2+ current. 3. The Q10 values (between 27 and 37 degrees C) for inhibition of exocytosis and the peak Ca2+ current amplitude were determined as > 5 and 1.6, respectively. From the temperature dependence of exocytosis, an energy of activation was estimated as 145 kJ mol‐1. 4. Suppression of exocytosis was not the result of a reduction of Ca2+ influx. When the Ca2+ currents were blocked by 30% (comparable to that produced by cooling) by using a low concentration of Co2+, exocytosis was reduced by < 25%. 5. Elevation of cytoplasmic free Ca2+, by photorelease of ‘caged’ Ca2+ from Ca(2+)‐nitrophenyl‐EGTA preloaded into the B‐cell, was equally effective at eliciting exocytosis at 24 and 34 degrees C. 6. Cooling produced 70% inhibition of exocytosis evoked by infusion of Ca2+ through the recording electrode. Omission of either MgATP or cAMP from the electrode solution resulted in a comparable reduction of exocytosis. Cooling had no additional inhibitory effect when exocytosis was already suppressed by removal of cytoplasmic MgATP. 7. Our data indicate that exocytosis of granules already docked beneath the membrane is little affected by cooling in the B‐cell. Instead, the high overall temperature sensitivity of insulin secretion arises because the replenishment of the readily releasable pool is temperature dependent.
Accumulation of intracellular HCO3‐ by Na(+)‐HCO3‐ cotransport in interlobular ducts from guinea‐pig pancreas.
Authors: Ishiguro H , Steward M C , Lindsay A R , Case R M
Published: December 2014

1. Short segments of interlobular duct were microdissected from guinea‐pig pancreas following enzymatic digestion. After overnight culture, intracellular pH (pH1) and Na+ concentration ([Na+]i) were measured by microfluorometry in duct cells loaded with either the pH‐sensitive fluoroprobe 2'7'‐bis(2‐carboxyethyl)‐5(6)‐carboxyfluorescein (BCECF) or the sodium‐binding benzofuran isophthalate (SBFI). 2. The transporters responsible for maintaining pHi above equilibrium were investigated by using the NH4Cl pulse technique to acid load the cells. In the absence of HCO3‐/CO2, the recovery of pH1 was Na+ dependent, abolished by 0.2 mM amiloride and by 10 microM N‐methyl‐N‐isobutylamiloride and was therefore attributed to Na(+)‐H+ exchange. 3. In the presence of HCO3‐/CO2, amiloride only partially inhibited the recovery from acid loading. The amiloride‐insensitive component was abolished by 0.5 mM H2DIDS and unaffected by depletion of intracellular Cl‐ and was therefore attributed to Na(+)‐HCO3‐ cotransport. 4. Stimulation with 10 nM secretin did not cause a significant change in pH1 despite a significant increase in HCO3‐ efflux. However, in the presence of secretin, addition of 0.5 mM H2DIDS caused a decline in pH1 that was three times more rapid than that obtained with 0.2 mM amiloride. 5. In secretin‐stimulated ducts, Na+ uptake increased when HCO3‐/CO2 was added to the bath and this increase was strongly inhibited by 0.5 mM H2DIDS. 6. We conclude that Na(+)‐HCO3‐ cotransport contributes approximately 75% of the HCO3‐ taken up by guinea‐pig pancreatic duct cells during stimulation with secretin. It is proposed that electrical coupling between HCO3‐ efflux at the luminal membrane and electrogenic Na(+)‐HCO3‐ cotransport at the basolateral membrane explains why secretin causes little change in pH1.
Electrotonic coupling between rat sympathetic preganglionic neurones in vitro.
Authors: Logan S D , Pickering A E , Gibson I C , Nolan M F , Spanswick D
Published: December 2014

1. Using the whole‐cell recording technique in rat spinal cord slices we have shown that 26% of sympathetic preganglionic neurones (SPNs) show spontaneous membrane potential oscillations. These oscillations consist of trains of biphasic waves, which we have termed spikelets because of their similarity to truncated action potentials. 2. The spikelets were inhibited by TTX and anaesthetics such as alpha‐chloralose but not by the intracellular application of lidocaine N‐ethyl bromide (QX‐314). 3. By stimulating the ventral roots we have demonstrated the presence of short‐latency depolarizations (SLDs) in oscillating neurones. These SLDs have a similar waveform to the spontaneous spikelets, and also show the ability to override the frequency of occurrence of the spontaneous spikelets. These observations suggest that the spikelets result from electrotonic coupling between the oscillating SPNs. 4. SLDs were also observed in a population of non‐oscillating, electrotonically coupled, quiescent SPNs. It was possible to induce oscillations in these neurones by the injection of depolarizing current (in the presence of QX‐314), suggesting that these neurones are also gap‐junction coupled. 5. Simultaneous whole‐cell recordings were obtained from twenty‐three pairs of SPNs. Two pairs displayed both spontaneous, synchronized oscillations and action potentials. Electrotonic coupling was confirmed by the detection of membrane polarization in both neurones in response to current injected into one neurone. In a further two pairs of quiescent SPNs, injection of depolarizing current pulses into one neurone induced action potential discharge in that neurone and a depolarization and oscillations in the other neurone. 6. The ability of groups of electrotonically coupled SPNs to generate spontaneous discharges within the spinal cord provides a novel mechanism for the integration and synchronization of information within the sympathetic nervous system.