bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanisms of action of acetylcholine in the guinea‐pig cerebral cortex in vitro.
Authors: McCormick D A , Prince D A
Published: December 2014

The mechanisms of action of acetylcholine (ACh) in the guinea‐pig neocortex were investigated using intracellular recordings from layer V pyramidal cells of the anterior cingulate cortical slice. At resting membrane potential (Vm = ‐80 to ‐70 mV), ACh application resulted in a barrage of excitatory and inhibitory post‐synaptic potentials (p.s.p.s) associated with a decrease in apparent input resistance (Ri). ACh, applied to pyramidal neurones depolarized to just below firing threshold (Vm = ‐65 to ‐55 mV), produced a short‐latency hyperpolarization concomitant with p.s.p.s and a decrease in Ri, followed by a long‐lasting (10 to greater than 60 s) depolarization and action potential generation. Both of these responses were also found in presumed pyramidal neurones of other cortical regions (sensorimotor and visual) and were blocked by muscarinic, but not nicotinic, antagonists. The ACh‐induced hyperpolarization possessed an average reversal potential of ‐75.8 mV, similar to that for the hyperpolarizing response to gamma‐aminobutyric acid (GABA; ‐72.4 mV) and for the i.p.s.p. generated by orthodromic stimulation (‐69.6 mV). This cholinergic inhibitory response could be elicited by ACh applications at significantly greater distance from the cell than the slow depolarizing response. Blockade of GABAergic synaptic transmission with solution containing Mn2+ and low Ca2+, or by local application of tetrodotoxin (TTX), bicuculline or picrotoxin, abolished the ACh‐induced inhibitory response but not the slow excitatory response. In TTX (or Mn2+, low Ca2+) the slow excitatory response possessed a minimum onset latency of 250 ms and was associated with a voltage‐dependent increase in Ri. Application of ACh caused short‐latency excitation associated with a decrease in Ri in eight neurones. The time course of this excitation was similar to that of the inhibition seen in pyramidal neurones. Seven of these neurones had action potentials with unusually brief durations, indicating that they were probably non‐pyramidal cells. ACh blocked the slow after‐hyperpolarization (a.h.p.) following a train of action potentials, occasionally reduced orthodromically evoked p.s.p.s, and had no effect on the width or maximum rate of rise or fall of the action potential. It is concluded that cholinergic inhibition of pyramidal neurones is mediated through a rapid muscarinic excitation of non‐pyramidal cells, resulting in the release of GABA. In pyramidal cells ACh causes a relatively slow blockade of both a voltage‐dependent hyperpolarizing conductance (M‐current) which is most active at depolarized membrane potentials, and the Ca2+‐activated K+ conductance underlying the a.h.p.(ABSTRACT TRUNCATED AT 400 WORDS)
Efferent discharges recorded from single skeletomotor and fusimotor fibres in man.
Authors: Ribot E , Roll J P , Vedel J P
Published: December 2014

Experiments were performed on awake human subjects in which single nerve fibre activity was recorded in the lateral peroneal nerve using tungsten micro‐electrodes as described by Hagbarth & Vallbo (1967, 1968a). The discharge of twelve single efferent fibres innervating the tibialis anterior muscle (t.a.) or the extensor digitorum longus muscle (e.d.l.) was recorded. On the basis of their functional activity, six fibres were identified as skeletomotor and six as fusimotor fibres. Skeletomotor fibres, which were completely silent in relaxed subjects, discharged when subjects performed voluntary isometric or isotonic contractions, they also fired during Jendrassik's manoeuvre and tonic vibration reflex (t.v.r.) induced by mechanical vibration applied to the distal muscle tendon. Units considered as fusimotor fibres were generally spontaneously active with some fluctuation in the discharge frequency. Various tests used to identify afferent fibres elicited no response of these fibres (nor of the skeletomotor fibres). Efferent fibres were considered as fusimotor because their discharges were uncorrelated with any activation of extrafusal muscle fibres. Several means were used to detect activation of extrafusal fibres: surface electromyogram (e.m.g.) electrodes, tungsten electrodes deeply implanted in the muscle and especially the use of a high‐sensitivity tension transducer (0 X 1 mN) placed on muscle tendons. The activity in fusimotor fibres could be either elicited or modulated under the following conditions: clenching of the fists, pinna twisting, mental computation, voluntary isometric contraction, passive phasic stretch of the muscle, environmental disturbances, subject laughing, the sound of hand clapping, and subject listening to manoeuvre instructions. Moreover, during spontaneous fusimotor fibre activity the subject was able to voluntarily stop the unit discharge. The results are compared to those obtained in animal studies and discussed with reference to the notion of alpha‐gamma linkage, static and dynamic gamma‐motoneurone activities, and to other available data concerning the effects of various stimulations on muscle spindle afferent activities in man.
Sodium and chloride transport by the tracheal epithelium of fetal, new‐born and adult sheep.
Authors: Olver R E , Robinson E J
Published: December 2014

In vitro measurements were made of Na+ and Cl‐ isotopic fluxes across the tracheal epithelium of mature fetal lambs (130‐143 days gestation), new‐born lambs (up to 41 days of age) and adult sheep under conditions of continuous short circuiting. The effects of a variety of drugs were examined, but only in the case of amiloride and isoprenaline were observations made in all three groups. Experiments designed to elucidate the mechanism of basal Cl‐ secretion were performed in adult trachea only. Under resting conditions the net flux of Na+ from lumen to submucosa exceeds that of Cl‐ in the reverse direction in fetal and adult trachea. In the new‐born the two fluxes are more or less equivalent in magnitude. In none of the three groups is the sum of ion fluxes significantly different from the short‐circuit current (Isc). Removal of Na+ from, or addition of furosemide (10(‐3) M) to, the solution bathing the submucosal surface of adult trachea has the effect of reducing Isc by an amount which approximates to the Cl‐ current (29%). At a concentration of 10(‐4) M on the submucosal side of adult trachea, ouabain causes potential difference and Isc to fall to zero within 70 min of addition to the bathing solution. Nevertheless, there remains a significant net Na+ flux from submucosa to lumen. The addition of isoprenaline (10(‐4) M) to the medium bathing the submucosal surface of both fetal and adult trachea causes an increase in the one‐way flux of Cl‐ from submucosa to lumen with consequent increase in net Cl‐ flux towards the lumen. (The Na+ fluxes are unchanged.) However, in the adult the Cl‐ secretory response to isoprenaline is very much less and is not accompanied by an increase in electrical conductance. As judged by the change in Isc, all the post‐natal fall in beta‐agonist responsiveness takes place within the 3 week period following birth. Whereas, in the fetus, the effect of luminally applied amiloride on the Na+ fluxes is negligible, in the adult the one‐way flux of Na+ from lumen to submucosa is reduced by 35% with a consequent 60% fall in net Na+ flux towards the submucosa.(ABSTRACT TRUNCATED AT 400 WORDS)
Electrophysiological effects of tetracaine in single guinea‐pig ventricular myocytes.
Authors: Carmeliet E , Morad M , Van der Heyden G , Vereecke J
Published: December 2014

The effect of tetracaine on the ionic current in enzymatically dissociated single guinea‐pig ventricular cells was studied using a two micro‐electrode voltage‐clamp technique. The myocytes were pre‐incubated with Cs+ and the experiments were performed at room temperature in order to reduce the contribution of the delayed outward current. Tetracaine decreased the maximum rate of rise of the action potential with a dissociation constant (KD) strongly dependent on the holding potential (0.77 microM at ‐80 mV, and 6.2 microM at ‐95 mV). Application of 20 microM‐tetracaine resulted in about a 50% reduction of the inwardly rectifying K+ current, while ten times higher concentrations were required to suppress the delayed K+ current. The inactivation time course of the Ca2+ current could be fitted with two exponentials, with time constants tau f = 15 ms and tau s = 150 ms at around 0 mV. Tetracaine decreased the amplitude of the Ca2+ current and speeded its decay. This effect was found to be primarily due to a marked inhibition of the amplitude of the slowly inactivating component (apparent KD = 80 microM, nH = 2). The drug had little effect on the time constants of the two components of Ca2+ channel inactivation. When Sr2+ or Ba2+ were the charge carriers, inactivation of the Ca2+ channel was again fitted with a fast and a slow exponential. In addition, a maintained (or very slowly inactivating) component was present. Tetracaine not only suppressed the amplitudes of the slowly inactivating and the maintained components, but also decreased the time constant of the slowly inactivating component. The results are consistent with a direct effect of tetracaine on the high threshold Ca2+ channel and do not support indirect effects of the drug secondary to suppression of Ca2+ release from internal stores.
Studies of capsule and capsular space of cat muscle spindles.
Authors: Fukami Y
Published: December 2014

The presence of hyaluronate in the capsular space of the cat muscle spindle was demonstrated using alcian blue staining at various pHs, the critical electrolyte concentration technique and hyaluronidase treatment. In spindles with intact capsules an extracellular marker, the dye Ruthenium Red, gained access to the capsular space through the gap in the sleeve region, but for a limited distance. In muscle spindles with the capsule nicked, the marker diffused into the capsular space in the equatorial region, revealing a dense network in this space which consisted of globular structures interconnected by thin filaments. Based on their thickness, these filaments were inferred to be hyaluronic acid, and the globular structures were inferred to be protein molecules. Longitudinal diffusion of the dye into the capsular space through the nicked site was limited. The limited diffusion is probably due to electrostatic binding of the dye, which is a hexavalent cation, to negatively charged glycosaminoglycan hyaluronate that is present in the space. The transcapsular potential was measured by use of glass micropipettes filled with 3 M‐KCl. The value was 15 mV +/‐ 4 (average +/‐ S.D., n = 12; range, 10‐20 mV) inside negative. The input resistance and capacitance of the capsule, measured with two independent electrodes, varied widely (1.3‐8.0 M omega and 0.5‐1.3 nF, n = 4) and the capsule showed marked delayed rectification to outward current pulses. [K+] in the space measured with K+‐sensitive resin‐filled glass micropipettes was a few millimolar higher than that in the bathing solution. The effects of [K+] and [Ca2+] on impulse activities were examined in spindles with intact capsules or with partially resected capsules. In spindles with intact capsules the effects of [K+] and [Ca2+] were significantly less or negligible compared with those in spindles with the capsule opened. Hyaluronidase (approximately 10(‐4) g/ml) added to the bathing solution around nicked capsules significantly reduced both resting and stretch‐induced impulse activities in 40‐50 min. By this time the capsular space was completely collapsed. An increase in [K+] of the bathing solution from 3.5 to 6 or 8 mM restored these impulse activities. A similar restoring effect was also observed when [Ca2+] in the bathing solution was reduced.(ABSTRACT TRUNCATED AT 400 WORDS)
Synaptic transmission in amphibian retinae during conditions unfavourable for calcium entry into presynaptic terminals.
Authors: Schwartz E A
Published: December 2014

Toad (Bufo marinus) retinae were peeled from the pigment epithelium and superfused over the photoreceptor surface with a calcium‐poor, cobalt‐rich medium. The shape of the electroretinogram indicated that post‐synaptic neurones received synaptic input. Adding the putative transmitters glutamate and N‐acetylhistidine changed the shape of the electroretinogram. The change suggests that an excess of the putative transmitters blocked a component of synaptic transmission that persisted when a retina was bathed in cobalt. Salamander (Ambystomatigrinum) retinae in hemisected eye cups were superfused over their vitreal surface. Intracellular responses were recorded from photoreceptors. Reducing the calcium concentration in the superfusing medium from 1 mM to less than 10 microM slowly changed responses produced by light. The change indicates that the calcium concentration in the extracellular space surrounding photoreceptors fell to less than 100 microM. When retinae were superfused with a medium containing 1 mM‐calcium, 3 mM‐barium, and 10 mM‐tetraethylammonium (TEA), rods produced action potentials that were later blocked by adding 1 mM‐cobalt. Blocking calcium channels with cobalt and lowering the extracellular calcium concentration should together block calcium‐dependent synaptic transmission. Intracellular responses were recorded from horizontal cells. After replacing external calcium with cobalt the membrane potential hyperpolarized and responses produced by light became smaller but did not entirely disappear. The responses that remained were less sensitive to light and had an altered shape. The change was reversible. Similar responses could be recorded after prolonged (30‐120 min) exposure to cobalt. Electrical synapses between horizontal cells were uncoupled by adding 10 microM‐forskolin to the cobalt medium. The polarity of a response could then be reversed if a cell was depolarized by injecting current. The observation of a reversal potential demonstrates that the response was produced by a conductance change. Intracellular responses were recorded from depolarizing and hyperpolarizing bipolar cells while retinae were superfused with cobalt‐rich medium. After changing to a cobalt‐free medium containing 1 mM‐calcium, responses produced by light were slightly smaller. Large responses were recorded after superfusing with cobalt‐rich, calcium‐poor medium for 30‐120 min. The results indicate that synaptic transmission by photoreceptors continues during conditions expected to block the entry of calcium into their presynaptic terminals.
Plasma expansion does not precipitate the fall in plasma vasopressin in humans drinking isotonic fluids.
Authors: Cotter T P , Gebruers E M , Hall W J , O'Sullivan M F
Published: December 2014

In a group of healthy humans, plasma vasopressin (AVP) levels fell on drinking either Tyrode or mannitol solutions isosmotic with plasma. Both the timing and magnitude of the fall were appropriate to account for the transient diuresis which followed the drinking. Although plasma expansion follows drinking Tyrode solution it occurred too late to account for the fall in plasma AVP. It was also too small to inhibit AVP secretion. Even though plasma volume tended to contract on drinking isosmotic mannitol solution a fall in plasma AVP and a diuresis occurred, similar to those found after drinking Tyrode solution. These findings appear to eliminate plasma volume expansion as the stimulus for the fall in plasma AVP and the associated diuresis on drinking isotonic fluids. In a further group of human subjects, bypassing the oropharynx by intragastric infusion resulted in a slower onset of diuresis after a water load. We suggest that receptors, as yet undefined, in the upper gastrointestinal tract contribute to the early stages of a water diuresis and account for the apparently inappropriate transient diuresis which follows the drinking of isotonic fluids.
Effects of forskolin on electrical behaviour of myenteric neurones in guinea‐pig small intestine.
Authors: Nemeth P R , Palmer J M , Wood J D , Zafirov D H
Published: December 2014

The actions of forskolin on electrical behaviour of myenteric neurones were investigated with intracellular recording methods in guinea‐pig small intestine. The actions of forskolin were: membrane depolarization, increased input resistance, suppression of post‐spike hyperpolarizing potentials and repetitive spike discharge. These effects occurred always in AH/Type 2 myenteric neurones and never in the cells classified as S/Type 1. Reversal potentials for the depolarizing effects were near the estimated potassium equilibrium potential. Analyses based on the 'constant field equation' indicated that the permeability ratios of K+ to other permeant ionic species were reduced by forskolin. Pretreatment of the neurones with a phosphodiesterase inhibitor potentiated the effects of forskolin. The results suggest that activation of adenylate cyclase by forskolin and subsequent elevation of intraneuronal adenosine 3',5'‐phosphate (cyclic AMP) mimic slow synaptic excitation in AH/Type 2 myenteric neurones. They support the possibility that cyclic AMP functions as a second messenger in signal transduction which appears to involve closure of calcium‐dependent K+ channels and other membrane changes that lead to depolarization and a dramatic increase in the excitability of the neurones.
Elevation of adenosine 3',5'‐phosphate mimics slow synaptic excitation in myenteric neurones of the guinea‐pig.
Authors: Palmer J M , Wood J D , Zafirov D H
Published: December 2014

Intracellular recording was used to assess the effects of elevation of intraneuronal adenosine 3',5'‐phosphate (cyclic AMP) on electrical behaviour of AH/Type 2 myenteric neurones in guinea‐pig small intestine. Intracellular injection of cyclic AMP resulted in membrane depolarization, increased input resistance, suppression of post‐spike hyperpolarizing potentials and enhanced excitability. Application of 8‐(4‐chlorophenylthio) cyclic AMP or 8‐bromo cyclic AMP, which are membrane permeant analogues of cyclic AMP, resulted in the same changes in electrical behaviour as intracellular injection of cyclic AMP. Treatment with the phosphodiesterase inhibitor, 3‐isobutyl‐l‐methylxanthine, resulted in the same changes in neuronal electrical behaviour as intracellular injection of cyclic AMP and application of permeable analogues of cyclic AMP. Each of the treatments, which were expected to elevate intraneuronal cyclic AMP, duplicated the electrophysiological behaviour of AH/Type 2 neurones during slow synaptic excitation. The results support the hypothesis that the biochemical basis for slow synaptic excitation in AH/Type 2 myenteric neurones of guinea‐pig small intestine is increased synthesis of cyclic AMP in the cell soma.
Intracellular calcium transients underlying the short‐term force‐interval relationship in ferret ventricular myocardium.
Authors: Wier W G , Yue D T
Published: December 2014

The influence of short‐term changes in stimulation pattern, both on the strength of contraction and on the amplitude of intracellular free‐Ca2+ transients, was investigated in ferret papillary muscles. Intracellular free‐Ca2+ concentration ([Ca2+]) was assessed from the luminescence emitted from muscles microinjected with the Ca2+‐sensitive photoprotein aequorin. The relationships between the strength of contraction and changes in stimulation pattern lasting 1‐2 beats could be described by monoexponential functions, all with very similar time constants (approximately 750 ms at 30 degrees C). Over the entire range that could be obtained, the strength of contraction, quantified by either peak tension or peak rate of tension development, was found to be linearly correlated with peak estimated [Ca2+]. Potential errors in the estimation of [Ca2+] from aequorin luminescence were analysed. To assess the influence of spatial non‐homogeneities of [Ca2+] on the estimate of [Ca2+], a model of Ca2+ diffusion in heart muscle was developed. The possible effect of using an inaccurate calibration curve was also examined. The results of these analyses indicate that [Ca2+] estimated from aequorin luminescence should be proportional to, if not equal to, true spatial average [Ca2+] (errors less than 7%). Given the conclusion of the analysis described above, it is inferred from points 2 and 3 that the relationships between peak spatial average [Ca2+] and short‐term changes in stimulation pattern are also represented by monoexponential functions, with time constants closely similar to those for the mechanical measurements. Exposure to ryanodine, a substance believed to inhibit the release of Ca2+ from sarcoplasmic reticulum, produced striking alterations in the pattern of variations in [Ca2+] mentioned above. These alterations are consistent with the hypothesis that the functions described above depend essentially upon properties of the sarcoplasmic reticulum.
Mucosal plexus and electrolyte transport across the rat colonic mucosa.
Authors: Bridges R J , Rack M , Rummel W , Schreiner J
Published: December 2014

Histological and functional studies were performed on a preparation of rat colonic mucosa from which the myenteric and submucosal plexus were removed. This preparation, referred to as the mucosa preparation, was used to investigate the potential influence of the mucosal plexus on electrolyte transport. Two neuropharmacologically active agents were used: sea anemone toxin (ATX II) to stimulate the fibres of the mucosal plexus and tetrodotoxin (TTX) to block the fibres of the mucosal plexus. The morphology of the neuronal network of the mucosal plexus was visualized after the epithelium was removed and whole mount preparations of the lamina propria and circular muscle layer of muscularis mucosae were stained histochemically for acetylcholinesterase activity. Several levels of organization within the mucosal plexus were seen. Each crypt is encircled by a thin bundle of fibres near the top. These thin fibres connect with thicker bundles of fibres that encircle groups of two to five crypts in a broad band. These bundles of fibres are in turn connected to larger bundles of fibres which lie in a flat plane just below the crypts along the circular muscle layer of muscularis mucosae. In addition perikarya and ganglia were revealed within the mucosal plexus. The base‐line net transport of Na+ and Cl‐ across the mucosa preparation was completely inhibited by ATX II (10(‐6) M). This effect of ATX II on net Na+ and Cl‐ transport was accompanied with an increase in the short‐circuit current (Isc), transmural conductance, and open‐circuit potential difference across the mucosa preparation. The effect of ATX II on Isc was dose dependent with a half‐maximal effective concentration at 5 X 10(‐8) M‐ATX II and a maximal effective concentration of 10(‐7) M. ATX II was effective only when added to the serosal solution. Net Na+ and Cl‐ transport was restored by TTX (10(‐6) M) to base‐line values in ATX II‐treated tissue. In addition the value of all three electrical parameters rapidly returned to the values measured before the addition of ATX II. TTX was effective in antagonizing the effects of ATX II only when added to the serosal solution. The results suggest that the regulation of electrolyte transport across the epithelium is at least one function of the mucosal plexus. Stimulation of the neurones within the mucosal plexus leads to the inhibition of electrolyte absorption.(ABSTRACT TRUNCATED AT 400 WORDS)
Human placental sulphate transport: studies on chorionic trophoblast brush border membrane vesicles.
Authors: Boyd C A , Shennan D B
Published: December 2014

SO4(2‐) transport by microvillous brush border membrane vesicles prepared from normal‐term human placenta has been studied using an ion‐exchange column assay. The uptake of this anion is time dependent and at 20 degrees C reaches a point of equilibrium by 60 min. 4,4'‐Diisothiocyanostilbene‐2,2'‐disulphonate (DIDS) and 4‐acetamido‐4'‐isothiocyanostilbene‐2‐2'‐disulphonate (SITS) (at 10(‐4) M) were found to inhibit approximately 80% of uptake. The concentration of DIDS producing half‐maximal inhibition was approximately 10(‐5) M. In contrast furosemide and probenecid were weak inhibitors of SO4(2‐) influx. The anions Cl‐, I‐, thiocyanate (SCN‐) and gluconate (cis‐side) caused minimal inhibition of SO4(2‐) influx. Salicylate (10 mM) produced 47% inhibition. The divalent anions thiosulphate, tungstate and unlabelled SO4(2‐) (10 mM) inhibited the uptake of SO4(2‐) (at 1 mM) to the same extent as DIDS. The vesicle membrane potential was altered by varying external K+ concentration using valinomycin. The DIDS‐sensitive SO4(2‐) influx was not affected by changes in membrane potential. A novel method has been developed for studying solute efflux from pre‐loaded vesicles. Both DIDS and a reduction in temperature were effective inhibitors of SO4(2‐) efflux. These results are discussed.
Venous mechanoreceptor input to neurones in the inferior mesenteric ganglion of the guinea‐pig.
Authors: Keef K D , Kreulen D L
Published: December 2014

Intracellular recordings were made in vitro from neurones of guinea‐pig inferior mesenteric ganglia (i.m.g.) attached to the mesenteric vasculature of the distal colon region. The inferior mesenteric vein was cannulated for distension. Non‐evoked continuous excitatory post‐synaptic potentials (e.p.s.p.s) and action potentials were recorded from 38% of neurones in the absence of the colon or any imposed perturbation. Continuous e.p.s.p.s were blocked by hexamethonium, tetrodotoxin and by transection of the lumbar colonic nerves. Distension of the inferior mesenteric vein altered the frequency of continuous activity in 23% of cells which exhibited continuous activity. Venous distension was associated with a depolarization in 31% of cells tested. The depolarization averaged 2.8 mV and in 89% of these was associated with an increase in membrane resistance. A further 14% of cells exhibited an increase in membrane resistance in the absence of depolarization. Venous distension increased the amplitude of fast e.p.s.p.s generated by stimulation of the lumbar splanchnic nerve or the intermesenteric nerve in 38% of cells tested. The results of this study demonstrate the existence of a mechanosensory pathway from the venous bed of the distal mesenteric region to the i.m.g. of the guinea‐pig and suggest that distension may enhance neural transmission by increasing the excitability of ganglionic cells.
Properties of the metallochromic dyes Arsenazo III, Antipyrylazo III and Azo1 in frog skeletal muscle fibres at rest.
Authors: Baylor S M , Hollingworth S , Hui C S , Quinta-Ferreira M E
Published: December 2014

Intact single twitch fibres from frog muscle were isolated and mounted in a normal Ringer solution (16 degrees C) on an optical bench apparatus for measuring fibre absorbance as a function of the wave‐length and polarization of the incident light. Fibre absorbance was measured in resting fibres both in the absence and in the presence of one of three metallochromic dyes: Arsenazo III, Antipyrylazo III and Azo1. In the absence of dye, the fibre intrinsic absorbance, Ai(lambda), measured as a function of wave‐length, lambda, was well described by the equation: Ai(lambda) = Ai(lambda long) (lambda long/lambda)X, where lambda long is a reference wave‐length selected to lie beyond the absorbance band of the dyes and X is the exponential index. For wave‐lengths between 480 and 810 nm, the average value of X was 1.1 for 0 deg polarized light (electric vector parallel to the fibre axis) and 1.3 for 90 deg polarized light (electric vector perpendicular to the fibre axis). The intrinsic absorbance at 0 deg, Ai,0(lambda), was somewhat larger than the intrinsic absorbance at 90 deg, Ai,90(lambda); for example, on average (n = 6), Ai,0 (810 nm) was 0.22, whereas Ai,90 (810 nm) was 0.016. Following dye injection, dye‐related absorbance was estimated from the measured total fibre absorbance by subtracting the component attributable to the intrinsic absorbance; additionally, for comparison with in vitro calibrations as a function of wave‐length, myoplasmic dye absorbance was corrected for the steady change in dye‐concentration with time that was attributable to dye diffusion. In fibres injected with either Arsenazo III or Antipyrylazo III, the dye‐related absorbance measured with 0 deg light, A0(lambda), was found to be significantly greater than that measured with 90 deg light, A90(lambda), indicating the presence of a resting 'dichroic' signal, A0(lambda)‐A90(lambda), attributable to bound and oriented dye molecules. On average, the lower limit estimated for the percentage of oriented dye was 2.8‐3.0% for Antipyrylazo III and 1.5‐1.8% for Arsenazo III, the population differences between the two dyes being statistically significant. The actual percentage of bound and oriented dye molecules is likely to be considerably larger for both dyes. For Arsenazo III, the wave‐length dependence of the dichroic signal was not distinguishably different from the 'isotropic' signal, defined as (A0(lambda) + 2A90(lambda))/3. which represents the average spectrum of all the dye molecules independent of orientation.(ABSTRACT TRUNCATED AT 400 WORDS)
The secondary spikes of climbing fibre responses recorded from Purkinje cell axons in cat cerebellum.
Authors: Campbell N C , Hesslow G
Published: December 2014

Responses evoked in Purkinje cells by climbing fibre activity were investigated by recording from Purkinje cell axons in the cerebellum of anaesthetized cats. Purkinje cell axons were identified by firing pattern and by latency of responses to stimulation of peripheral nerve and of the inferior olive. Axonal climbing fibre responses usually consisted of one to two spikes, suggesting that normally only the initial spike or, at most, this and one of the secondary spikes are propagated down the Purkinje cell axon. When two successive climbing fibre responses were evoked, the number of spikes in the second response was increased, usually up to three to five. This effect could be obtained at stimulation intervals of up to 100 ms. In a few cases it was possible for a climbing fibre response to be preceded by a parallel fibre volley evoked by stimulation of the cerebellar surface. This increased the number of spikes in the axonal climbing fibre response. The results suggest that the number of propagated spikes in the climbing fibre response can be modified by a preceding input to the Purkinje cell.
Dynorphin A decreases voltage‐dependent calcium conductance of mouse dorsal root ganglion neurones.
Authors: Macdonald R L , Werz M A
Published: December 2014

The actions of the opioid peptides dynorphin A and (Leu)enkephalin were assessed on calcium‐dependent action potentials and inward calcium currents recorded from somata of mouse dorsal root ganglion (d.r.g.) neurones grown in primary dissociated cell culture. Dynorphin A and (Leu)enkephalin decreased the duration of somatic calcium‐dependent action potentials in a portion of d.r.g. neurones impaled with potassium acetate‐filled micropipettes. When substantial potassium conductance was blocked by intracellular injection of caesium acetate, d.r.g. neurones continued to respond to dynorphin A but responses to (Leu)enkephalin were abolished. In voltage‐clamp experiments, dynorphin A but not (Leu)enkephalin reduced the magnitude of inward calcium currents. Dynorphin A responses were blocked by the opiate antagonist naloxone. The dynorphin A effect was due to reduction of voltage‐dependent calcium conductance since dynorphin A reduced depolarization‐evoked inward currents but did not alter membrane conductance following blockade of calcium channels by cadmium, and because dynorphin A reduced the instantaneous current‐voltage slope (chord conductance) during step commands that produced maximal activation of voltage‐dependent calcium conductance. Dynorphin A binds with high affinity to kappa‐opioid receptors. (Leu)enkephalin, which has affinity for both mu‐ and delta‐receptors but not for kappa‐opioid receptors, was without effect on calcium conductance. Therefore, we suggest that kappa‐receptors are coupled to voltage‐dependent calcium‐channels and that binding of dynorphin A produces a decrease of calcium current.
Sensitization of aortic and cardiac baroreceptors by arginine vasopressin in mammals.
Authors: Abboud F M , Aylward P E , Floras J S , Gupta B N
Published: December 2014

Vasopressin facilitates the baroreflex control of the circulation. The peptide may act at several sites to augment the baroreflex. In this study we examined the effect of vasopressin on aortic baroreceptors in anaesthetized rabbits and on left ventricular mechanoreceptors in anaesthetized cats. Vasopressin (16 mu./kg. min) did not change resting nerve discharge in single fibres from aortic baroreceptors. Vasopressin (16 mu./kg. min) significantly enhanced the response of single aortic nerve fibre discharge to elevation in arterial pressure. The slope relating nerve activity to mean arterial pressure increased from 0.24 +/‐ 0.05 (mean +/‐ S.E. of mean) to 0.50 +/‐ 0.16 impulses/cardiac cycle. mmHg (P less than 0.05) in ten aortic medullated fibres and from 0.06 +/‐ 0.03 to 0.18 +/‐ 0.04 impulses/cardiac cycle. mmHg (P less than 0.05) in three non‐medullated fibres. Vasopressin (16 mu./kg. min) did not change resting nerve discharge in single fibres from left ventricular mechanoreceptors. Vasopressin (16 mu./kg. min) significantly enhanced the response of single nerve fibre discharge from left ventricular mechanoreceptors in response to elevation of left ventricular end‐diastolic pressure. The slope relating nerve activity to left ventricular end‐diastolic pressure increased from 0.24 +/‐ 0.07 to 0.32 +/‐ 0.07 impulses/cardiac cycle. mmHg (P less than 0.05) in six medullated fibres and from 0.10 +/‐ 0.01 to 0.15 +/‐ 0.02 impulses/cardiac cycle. mmHg (P less than 0.05) in four non‐medullated fibres. These data show that vasopressin sensitizes high and low pressure baroreceptors and suggest a mechanism by which vasopressin may facilitate the baroreflex control of the circulation.
Factors affecting the expression of acetylcholine receptors on rat sensory neurones in culture.
Authors: Cooper E , Lau M
Published: December 2014

Sensory neurones from nodose ganglia of new‐born rats were grown in dissociated tissue culture either with or without satellite cells. When cultured without satellite cells, most neurones developed sensitivity to acetylcholine (ACh); time‐course experiments indicated that the neurones acquire their sensitivity during the second to third week in culture. Most neurones co‐cultured with satellite cells did not develop ACh sensitivity. Delayed removal of satellite cells 8‐12 days after plating resulted in few neurones acquiring ACh sensitivity. Delayed addition of satellite cells to neuronal cultures that were initially grown without satellite cells had no effect on the number of ACh‐sensitive neurones. The potential to develop ACh sensitivity in culture without satellite cells decreases with the age of the neurones at the time of culturing; few neurones from 2‐week‐old animals developed sensitivity to ACh when cultured without satellite cells. These results indicate that there is some influence from satellite cells that prevents nodose neurones from developing ACh sensitivity in culture and suggests that this influence may also operate in vivo.
Electrophysiology and morphology of vasoactive‐intestinal‐peptide‐immunoreactive neurones of the guinea‐pig ileum.
Authors: Katayama Y , Lees G M , Pearson G T
Published: December 2014

Simultaneous intracellular staining and electrophysiological recording techniques have been applied to neurones of guinea‐pig myenteric plexus‐longitudinal muscle preparations. With micro‐electrodes filled with a solution of the fluorescent dye Lucifer Yellow, neurones were first characterized morphologically and electrophysiologically, and subsequently subjected to an indirect immunohistochemical method for the detection of vasoactive intestinal peptide (VIP)‐like immunoreactivity. Cross‐correlations of morphology, electrophysiology and VIP immunoreactivity were successfully achieved in a total of 164 neurones. Sixty‐three had the slow after‐hyperpolarization characteristic of AH neurones; 101 cells displayed fast excitatory post‐synaptic potentials (e.p.s.p.s) in response to transmural or focal stimulation and were therefore, by definition, S neurones. Unequivocal VIP immunoreactivity was observed in 25 (25%) S neurones, which, with only one exception, had Dogiel Type I morphology (i.e. many short soma processes and a single long process). In contrast, AH neurones had Dogiel Type II morphology (i.e. smooth soma with several long processes) and none showed VIP immunoreactivity. In addition to cholinergic fast e.p.s.p.s., non‐cholinergic slow synaptic inputs were evoked in seventeen of the twenty‐two VIP‐immunoreactive S neurones tested. Both the fast and slow e.p.s.p.s could be elicited by stimulation of the preparation, oral or aboral to the site of recording. These observations demonstrate that, in the guinea‐pig ileum, myenteric plexus neurones showing VIP immunoreactivity are of a single electrophysiological type (S neurones) and belong to essentially one morphological class (Dogiel Type I).
An enzymatic mechanism for calcium current inactivation in dialysed Helix neurones.
Authors: Chad J E , Eckert R
Published: December 2014

'Wash‐out' and inactivation of the Ca current were examined in dialysed, voltage‐clamped neurones of Helix aspersa under conditions that isolate the Ca current virtually free of other currents. EGTA or other internal Ca2+ chelators were routinely omitted from the dialysate. The time‐dependent loss, or wash‐out, of Ca current was slowed by addition to the dialysing solution of agents, such as dibutyryl adenosine 3'‐5'‐cyclic monophosphate (dibutyryl cyclic AMP), Mg adenosine 5'‐triphosphate (ATP) and the catalytic subunit of cyclic‐AMP‐dependent protein kinase, that promote protein phosphorylation and by EGTA. However, neither the phosphorylation‐promoting agents nor internal EGTA prevented wash‐out entirely, nor did they significantly restore previously 'washed‐out' current. With phosphorylating agents in the dialysing solution, the irreversible development of wash‐out was greatly reduced by introduction of leupeptin, an inhibitor of protease activity. Thus, the irreversible component of wash‐out appears to result from a Ca‐dependent proteolytic process. In the presence of leupeptin alone, Ca current amplitude continued to decline: however, the current could be largely or fully restored with addition of catalytic subunit, dibutyryl cyclic AMP, and Mg ATP to the dialysing solution. Thus, inhibition of proteolysis revealed a reversible component of wash‐out that appears to result from dephosphorylation. During perfusion with leupeptin, Mg ATP, dibutyryl cyclic AMP and catalytic subunit the Ca current remained stable for up to several hours without addition of internal Ca2+ buffer. The rate of inactivation of the current that occurs during a depolarizing step showed only a very gradual decline during this time. Under these conditions, perfusion with calcineurin, a Ca‐calmodulin‐dependent phosphatase, caused a significant increase in the rate of Ca current inactivation. This inactivation was virtually eliminated by introduction of EGTA or by replacement of external Ca2+ with Ba2+, which is consistent with the ion dependency for calmodulin‐dependent activation of calcineurin. When ATP in the dialysate was replaced with ATP‐gamma‐S (adenosine 5'‐O‐(thiotriphosphate], an analogue that donates a thiophosphate group resistant to hydrolysis, the rate of inactivation slowed. Since Ca‐dependent inactivation during step depolarizations is enhanced by conditions that promote dephosphorylation, and Ca current wash‐out is slowed by conditions that promote phosphorylation, inactivation and reversible wash‐out appear to be related.(ABSTRACT TRUNCATED AT 400 WORDS)
Quantitative description of three modes of activity of fast chloride channels from rat skeletal muscle.
Authors: Blatz A L , Magleby K L
Published: December 2014

The steady‐state kinetic properties of single Cl‐ channels with fast kinetics active at resting membrane potentials in cultured rat skeletal muscle were studied using the patch‐clamp technique. Membrane patches containing single active Cl‐ channels were often observed, and binomial analysis of the percentage open time in membrane patches containing several Cl‐ channels indicated that the channels did not occur as obligatory dimers and that they gated independently of one another. Channel activity could be divided into three categories: normal, which included about 99% of the openings and closings; buzz mode, which included about 1% and consisted of bursts of about 50 brief open and shut intervals; and inactivated shut states which included about 0.01% of the shut intervals and lasted for seconds, and occasionally minutes. The method of maximum likelihood was used to determine the number of significant exponential components required to fit the distributions of open and shut intervals during normal activity. Open interval distributions required at least two components, with time constants of 0.52 and 1.5 ms at ‐40 mV and 7.6 degrees C. Shut interval distributions required at least five exponential components, with time constants of 0.064, 0.72, 1.9, 12.3 and 350 ms. Kinetic reaction schemes were developed for the normal and buzz mode using maximum likelihood techniques to determine the most likely models and rate constants. In developing these models the effects of limited time resolution and missed events were taken into account. Each model tested typically had two or more sets of equally likely rate constants. Incorrect sets of rate constants resulting from the effect of missed events could be eliminated by analysis of the data with different time resolutions. Normal activity could be accounted for by several different seven‐state models with two open and five shut states. As different models could be found that gave identical descriptions of the data, the distributions of open and shut intervals were not sufficient to define a unique model. It was established that no other seven‐state models would be found that describe the distributions of open and shut intervals during normal activity better than the most likely presented models.(ABSTRACT TRUNCATED AT 400 WORDS)
Rapid resetting of rabbit aortic baroreceptors and reflex heart rate responses by directional changes in blood pressure.
Authors: Burke S L , Dorward P K , Korner P I
Published: December 2014

In both anaesthetized and conscious rabbits, perivascular balloon inflations slowly raised or lowered mean arterial pressure (M.A.P.), at 1‐2 mmHg/s, from resting to various plateau pressures. Deflations then returned the M.A.P. to resting. 'Steady‐state' curves relating M.A.P. to unitary aortic baroreceptor firing, integrated aortic nerve activity and heart rate were derived during the primary and return pressure changes and they formed typical hysteresis loops. In single units, return M.A.P.‐frequency curves were shifted in the same direction as the primary pressure changes by an average 0.37 mmHg per mmHg change in M.A.P. Shifts were linearly related to the changes in M.A.P. between resting and plateau levels for all pressure rises and for falls less than 30 mmHg. They were established within 30 s and were quantitatively similar to the rapid resetting of baroreceptor function curves found 15 min‐2 h after a change in resting M.A.P. (Dorward, Andresen, Burke, Oliver & Korner, 1982). Unit threshold pressures were shifted within 20 s to the same extent as the over‐all curve shift to which they contributed. In the whole aortic nerve, return M.A.P.‐integrated activity curves were shifted to same degree as unit function curves in both anaesthetized and conscious rabbits. Simultaneous shifts of return reflex M.A.P.‐heart rate curves were also seen in conscious rabbits within 30 s. During M.A.P. falls, receptor and reflex hysteresis was similar, but during M.A.P. rises, reflex shifts were double baroreceptor shifts, suggesting the involvement of other pressure‐sensitive receptors. We conclude that hysteresis shifts in baroreceptor function curves, which follow the reversal of slow ramp changes in blood pressure are a form of rapid resetting. They are accompanied by rapid resetting of reflex heart rate responses. We regard this as an important mechanism in blood pressure control which produces relatively high‐gain reflex responses, during slow directional pressure changes, over a wider range of absolute pressure levels than would otherwise be possible.
'Concentration‐clamp' study of gamma‐aminobutyric‐acid‐induced chloride current kinetics in frog sensory neurones.
Authors: Akaike N , Inoue M , Krishtal O A
Published: December 2014

Kinetics of the activation and desensitization phases of gamma‐aminobutyric acid (GABA)‐induced Cl‐ current (ICl) were studied in single frog sensory neurones using the 'concentration‐clamp' technique which enables perfusion of drugs with the time constant of about 3 ms. Both activation and desensitization phases of GABA response consisted of a single exponential at low concentrations and a double exponential at high concentrations. The time constant of the fast kinetic component in each phase was relatively stable, about 5 ms for activation and 3 s for desensitization over concentrations from 3 X 10(‐5) to 3 X 10(‐4) M, whereas those of the slow kinetic component decreased with increasing concentrations. The two kinetic components in both phases showed the same reversal potential. The slow and fast activation components recovered sensitivity from desensitization with different time courses: the recovery rate of the fast activation component was slow and that of the slow one, rapid. The peak ICl elicited at GABA concentrations below 10(‐5) M increased disproportionally at more negative membrane potentials, thereby suggesting that the activation kinetics is voltage dependent. The steady‐state ICl‐voltage relationship obtained with less than 10(‐5) M‐GABA showed a non‐linearity, probably due to voltage dependence of activation rather than that of desensitization kinetics. These results suggest the presence of at least two different GABA receptor‐Cl‐ ionophore complexes with a different affinity and kinetics.
Effects of high potassium on the release of [3H]dopamine from the cat carotid body in vitro.
Authors: Almaraz L , Gonzalez C , Obeso A
Published: December 2014

Using an in vitro preparation of the cat carotid body, we have characterized the release of [3H]dopamine (DA) induced by high extracellular K+. Pulse superfusion (3 min) with high K+ Tyrode solution (20‐80 mM) evoked a concentration‐dependent release of [3H]DA from type I cells with a threshold at about 20 mM‐extracellular K+ and a plateau at about 60 mM‐extracellular K+. Equivalent low extracellular Na+ concentration ([Na+]o) solutions osmotically balanced with sucrose did not induce release. The high extracellular K+ concentration ([K+]o)‐evoked release of [3H]DA by type I cells was dependent on the presence of Ca2+ in the superfusion media. On prolonged (10‐14 min) superfusion with high K+ Tyrode solution, the [3H]DA release evoked by 60 mM‐extracellular K+ was transient, while that evoked by 30 mM‐extracellular K+ was sustained. In preparations superfused for 6 min with 60 mM‐extracellular K+ and zero extracellular Ca2+ concentration ([Ca2+]o) Tyrode solution, reintroduction of Ca2+ did not elicit a secretory response. Ba2+ was a potent secretagogue of [3H]DA in preparations superfused with normal and zero [Ca2+]o Tyrode solution. Additionally, Ba2+ was capable of eliciting a secretory response from type I cells in preparations previously exposed (6 min) to 60 mM‐extracellular K+, whether or not [Ca2+]o was present. With regards to stimulus‐secretion coupling, our results indicate that high [K+]o probably depolarizes type I cells. This effect would, in turn, activate voltage‐dependent Ca2+ channels, allowing the entrance of this ion to activate the neurosecretory response.