Concentration Clamp Research Articles

Acute alcohol does not impair attentional inhibition as measured with Stroop interference scores but impairs Stroop performance

RationaleInhibition is a core executive function and refers to the ability to deliberately suppress attention, behavior, thoughts, and/or emotions and instead act in a specific manner. While acute alcohol exposure has been shown to impair response inhibition in the stop-signal and Go/NoGo tasks, reported alcohol effects on attentional inhibition in the Stroop task are inconsistent. Notably, studies have operationalized attentional inhibition variably and there has been intra- and inter-individual variability in alcohol exposure.ObjectiveThis study aimed to examine the acute effects of alcohol on attentional inhibition, considering previous limitations.MethodsIn a single-blind, cross-over design, 40 non-dependent participants with a medium-to-high risk drinking behavior performed a Counting Stroop task (CST) under a baseline and an arterial blood alcohol concentration (aBAC) clamp at 80 mg%. Attentional inhibition was assessed as the alteration of reaction times (RT), error rates (ER), and inverse efficiency scores (IES) between incongruent and congruent trials (interference score). Stroop performance was also assessed regardless of trial-type.ResultsCompared to saline, acute alcohol exposure via an aBAC clamp did not affect CST interference scores but increased RTs and IES in both incongruent and congruent trials.ConclusionsAttentional inhibition (Stroop interference score) was not impaired by clamped moderate alcohol exposure. Acute alcohol impaired Stroop performance evidenced by a general increase in response times. Our findings suggest that response and attentional inhibition do not share the same neurocognitive mechanisms and are affected differently by alcohol. Results could also be explained by automated behaviors known to be relatively unaffected by acute alcohol.

Extracellular cAMP inhibits P2X3 receptors in rat sensory neurones through G protein‐mediated mechanism

To identify the mechanisms of P2X(3) receptor inhibition by extracellular cyclic adenosine monophosphate (cAMP) in rat dorsal root ganglion (DRG) neurones. Whole-cell currents were measured in cultured DRG neurones using the combination of voltage and concentration clamp. We have found that extracellular cAMP inhibits P2X(3)-mediated currents in a concentration- and use-dependent manner. The P2X(3) currents, activated by ATP applied every 4 min, were inhibited by 55% in the presence of 10 microm cAMP and by 81% in the presence of 30 microm cAMP. At 8 min interval between ATP applications the same concentration of cAMP did not alter the currents. Addition of 0.5 mm of guanosine 5'-O-(2-thiodiphosphate) to intracellular solution blocked the inhibitory action of cAMP. The inhibitory effects of cAMP were not mimicked by extracellular application of 30 mum adenosine. In this paper, we demonstrate, for the first time, that extracellular application of cAMP to rat sensory neurones inhibits P2X(3) receptors via a G protein-coupled mechanism in a use-dependent manner, thus indicating the neuronal expression of specific plasmalemmal cAMP receptor.

Lack of P2X receptor mediated currents in astrocytes and GluR type glial cells of the hippocampal CA1 region

Purinergic signalling plays a major role in intercellular communication between neurons and glial cells. Glial cells express metabotropic receptors for ATP and adenosine, the latter being activated after ATP cleavage through extracellular ecto-ATPase activity. Ionotropic receptors for extracellular ATP, so called P2X receptors, might contribute to neuron-glia signalling. However, experimental evidence for the presence of these receptors in glial cells is less convincing so far. In a previous study, immunohistochemistry was used to identify P2X(1-4,6,7) receptor protein in S100beta-positive hippocampal glial cells. Applying patch clamp and fast application techniques, here we challenged the question of the functional expression of these receptors. Time correlated membrane currents served as test criterion for receptor function, since P2X receptor activation leads to the opening of unspecific cation channels in a millisecond time scale. Agonists were applied via short pressure puffs, with a fast concentration clamp method and through UV flash triggered photolysis of caged ATP. Two types of murine hippocampal macroglial cells, both labelled by the expression of green fluorescence protein driven by the human glial fibrillary acid protein promoter, were analysed in acute brain slices and in freshly dissociated cell suspensions. Surprisingly, ATP or related agonists completely failed to activate currents. Additionally, changes in spontaneously occurring glial postsynaptic currents were never observed. These results have been verified using rat and human hippocampal tissue as well as investigating cells from P2X7 knock out mice. It is concluded that in acute preparations, astroglial cells of the hippocampal CA1 subfield do not express functional P2X receptors.

Improved transformation of morphometric measurements for a priori parameter estimation in a physiologically-based pharmacokinetic model of ethanol

Prescription of the brain's time course of exposure to experimentally administered ethanol can be achieved with intravenous infusion profiles computed from a physiologically-based pharmacokinetic (PBPK) model of alcohol distribution and elimination. Previous parameter estimation employed transformations of an individual's age, height, weight and gender inferred from the literature, with modeling errors overcome with real-time, intermittent feedback. Current research applications, such as ethanol exposures administered during fMRI scanning, require open-loop infusions, thus improved transformation of morphometric measurements. Records of human breath alcohol concentration (BrAC) clamp experiments were analyzed. Optimal, unique PBPK parameters of a model of the distribution and elimination of ethanol were determined for each record and found to be in concordance with parameter values published by other investigators. A linear transformation between the readily measurable physical characteristics or morphometrics, including gender, age, height, weight, and TBW estimates, and the model parameters were then determined in a least squares sense according to the formula θ = F( x) = F m x where x = (age height weight TBW) T ∈ R 4 and θ = ( R C V P V B m max k AT) T ∈ R 5. The transformation was then evaluated with several parameter prediction performance measures. A substantial improvement in all error statistics, in relation to an earlier affine transformation that used only body weight as the relevant morphometric was obtained. Deviation from the measured response was reduced from 27 to 20%. Error in parameter estimation was reduced from 109 to 38%. Percent alcohol provided in error was reduced from 46 to 28%. Error in infusion profile estimation was reduced from 55 to 33%. The algorithm described, which optimizes individual pharmacokinetic parameter values and then subsequent extension to a priori prediction, while not unique, can be readily be adapted to other molecules and pharmacokinetic models. This includes those used for distinct purposes, such as automated control of anesthetic agents.

Contribution of the cytoskeleton and the phospholipase C signaling pathway to fluid stream-induced membrane currents

A fluid stream induced by a concentration clamp system evokes in Xenopus oocytes a deformation of the membrane which results in transient chloride currents of high amplitude (stream-evoked inward current, I i,st) during calcium-activated chloride current oscillations. The involvement of cytoskeleton elements and of components of the phospholipase C-dependent signaling pathway on the generation of the I i,st were investigated. Incubation of the oocytes with cytoskeleton-disrupting agents exerted no effects on generation of the I i,st, suggesting that the mechanotransduction is not mediated by these structures. The fluid stream induced an elevation of the submembraneous calcium concentration, as measured by an increase of Fluo-4-mediated fluorescence after the stimulus. Lowering the intracellular calcium concentration by injection of calcium chelators or depleting inositol 1,4,5-triphosphate (InsP 3)-sensitive calcium stores by blockers of the calcium pumps suppressed the generation of the I i,st in most cases. Furthermore, the phospholipase C inhibitor U73122 reversibly blocked the I i,st. The results suggest that the fluid stream leads to a membrane stretch which modulates directly or indirectly the activity of a membrane-bound phospholipase C. The phospholipase C transiently elevates the InsP 3 concentration, in turn releasing calcium from InsP 3-sensitive internal calcium stores, thus evoking an enhanced calcium-sensitive chloride current.

Inotropic effect of digoxin in humans: mechanistic pharmacokinetic/pharmacodynamic model based on slow receptor binding.

The purpose of this study was to construct a mechanistic pharmacokinetic/pharmacodynamic (PK/PD) model for digoxin that describes the relationship between plasma concentration and inotropic response. On the basis of results obtained in the isolated perfused rat heart, a PK/PD model for digoxin in humans was developed. In fitting the model to previously published bolus dose and concentration clamp data (shortening of electromechanical systole), the plasma concentration-time curves were used as forcing functions in the computer program ADAPT II. The mechanistic approach allowed a modeling of digoxin pharmacodynamics which is consistent with available inotropic response data. The estimates of the receptor binding parameters were in the same order of magnitude as those measured in vitro for ouabain. The mechanistic model explained the parameters of the empirical link model (EC50, Emax and delay time tau) in terms of the underlying processes, suggesting that the long equilibration half-time of 13 h is due to slow receptor binding. The empirical link model, in contrast, is not compatible with a noninstantaneous receptor binding process and led to estimates of the delay time tau that were dependent on the digoxin administration schedule. The new, mechanistic model may provide a rationale for better understanding of digoxin pharmacodynamics and could become a tool to bridge the gap between in vitro and in vivo studies.

Self‐Reported Subjective Perception of Intoxication Reflects Family History of Alcoholism When Breath Alcohol Levels Are Constant

Background The premise of this study is that the increased familial risk for alcoholism is associated with genetic determinants of the response to alcohol, characterized by sensitivity and adaptation. Following a single administration, sensitivity is the initial response to alcohol, expressed as the change in dependent measures from baseline. Adaptation of dependent measures within a single exposure to alcohol can be expressed as acute tolerance (recovery of dependent measures toward baseline values) or sensitization (movement of dependent measure further away from baseline values). This study tested the hypothesis that family history‐positive (FHP) subjects are more sensitive and more adaptive to alcohol compared with family history‐negative (FHN) subjects.Methods The initial response and development of adaptation to alcohol were assessed by using self‐reported subjective perceptions during a breath alcohol concentration (BrAC) clamp of 60 mg%. The Biphasic Alcohol Effects Scale, the Sensation Scale and a visual analog scale of intoxication were acquired at baseline, after the BrAC clamp was established, and after maintenance of the clamp for 105 min.Results FHP subjects were more sensitive to alcohol compared with FHNs, as evidenced by greater changes in feelings of intoxication when the BrAC clamp was initially achieved. While the clamp was maintained, the FHP subjects adapted to the effects of alcohol and their perceptions of intoxication became indistinguishable from those of the FHN subjects. The FHP subjects had developed acute tolerance to alcohol, whereas the FHN subjects did not. Other self‐reported perceptions of alcohol's effects did not distinguish between the groups.Conclusions A differential family history of alcoholism was reflected in self‐reported subjective perceptions of intoxication when the brain's exposure to a specified concentration of alcohol was held constant (BrAC of 60 mg%). FHP subjects reported greater intoxication after alcohol and subsequently developed acute tolerance to alcohol compared with FHN subjects.

Self-Reported Subjective Perception of Intoxication Reflects Family History of Alcoholism When Breath Alcohol Levels Are Constant

Background The premise of this study is that the increased familial risk for alcoholism is associated with genetic determinants of the response to alcohol, characterized by sensitivity and adaptation. Following a single administration, sensitivity is the initial response to alcohol, expressed as the change in dependent measures from baseline. Adaptation of dependent measures within a single exposure to alcohol can be expressed as acute tolerance (recovery of dependent measures toward baseline values) or sensitization (movement of dependent measure further away from baseline values). This study tested the hypothesis that family history-positive (FHP) subjects are more sensitive and more adaptive to alcohol compared with family history-negative (FHN) subjects. Methods The initial response and development of adaptation to alcohol were assessed by using self-reported subjective perceptions during a breath alcohol concentration (BrAC) clamp of 60 mg%. The Biphasic Alcohol Effects Scale, the Sensation Scale and a visual analog scale of intoxication were acquired at baseline, after the BrAC clamp was established, and after maintenance of the clamp for 105 min. Results FHP subjects were more sensitive to alcohol compared with FHNs, as evidenced by greater changes in feelings of intoxication when the BrAC clamp was initially achieved. While the clamp was maintained, the FHP subjects adapted to the effects of alcohol and their perceptions of intoxication became indistinguishable from those of the FHN subjects. The FHP subjects had developed acute tolerance to alcohol, whereas the FHN subjects did not. Other self-reported perceptions of alcohol's effects did not distinguish between the groups. Conclusions A differential family history of alcoholism was reflected in self-reported subjective perceptions of intoxication when the brain's exposure to a specified concentration of alcohol was held constant (BrAC of 60 mg%). FHP subjects reported greater intoxication after alcohol and subsequently developed acute tolerance to alcohol compared with FHN subjects.

BN52021, a platelet activating factor antagonist, is a selective blocker of glycine-gated chloride channel

We have found that the platelet activating factor antagonist (BN52021) is an effective blocker of the glycine (Gly) receptor-mediated responses in the hippocampal pyramidal neurons of rat. Using the whole-cell voltage clamp and concentration clamp recording techniques, we investigated the mechanism underlying the inhibitory action of this terpenoid on the glycine-induced chloride current. BN52021 selectively and reversibly inhibits glycine current in a non-competitive and voltage-dependent fashion. The antagonistic effect of this substance is more pronounced at positive membrane potentials. At holding potential −70 mV and in the presence of 200 μM glycine IC 50 value for the blocking action of BN52021 was 270±10 nM. Repetitive applications of BN52021 reveal the use-dependence of its blocking action. When co-applied with strychnine (STR), a competitive glycine receptor antagonist, BN52021 does not alter the IC 50 value for strychnine. The inhibitory effect of BN52021 on gamma-aminobutyric acid (GABA) current is at least 25 times less potent than the effect on glycine current. This substance fails to affect AMPA and NMDA responses. It may be concluded that BN52021 inhibits glycine-gated Cl − channels by interacting with the pore region and does not compete for the strychnine-binding centre.

Modulation of ion channels in rat neurons by the constituents of Hypericum perforatum.

Despite almost forty years of widespread use of antidepressant drugs, their mode of action is still unknown. Hyperforin, a phloroglucinol derivative, is a major pharmacologically and therapeutically active constituent of Hypericum perforatum extract that is widely used as an herbal antidepressant drug. However, the mechanism or mechanisms of action of these naturally abundant, non-toxic extracts remain unclear. Enzymatically isolated patch-clamped rat central and peripheral neurons exposed to rapid changes in the composition of external medium (concentration clamp) were used in our experiments to investigate the modulation of the various voltage- and ligand-gated channels by hyperforin, as well as by other constituents of Hypericum perforatum. At nanomolar concentrations, hyperforin induced significant inhibition of various ion channels. In the case of P-type Ca2+ channels, we established that hyperforin acts via interaction with calmodulin or through calmodulin-activated pathways involving at least one second messenger. The results presented here indicate that multiple mechanisms and extract constituents may be involved in the antidepressant action of Hypericum extracts, and that they could also possess neuroprotective and analgesic effects.

The neurotoxicity of environmental pollutants: the effects of tin (Sn2+) on acetylcholine-induced currents in greater pond snail neurons.

Inorganic and organic tin compounds present in aqueous ecosystems have diverse effects on the behavior of living organisms. With the aim of identifying possible correlates of these actions, we studied the effects of both types of Sn2+. The effects of SnCl2 and Sn(CH3)2 on acetylcholine-activate currents were studied on identified neurons of the mollusk Lymnaea stagnalis L. using a two-microelectrode membrane potential clamping technique and by intracellular dialysis with potential and ion concentration clamping. Experiments were performed on single neurons after isolation and on whole ganglion preparations. SnCl2 decreased acetylcholine-induced influx currents; the effect was dose-dependent. The effective threshold concentration, measured by the two-microelectrode membrane potential clamping method, was 0.1 microM, with saturation occurring at 5 microM SnCl2. After a 10-min preapplication of SnCl2, the effect was stronger (20%) than after treatment for 3 min (7%). Similar results were obtained after application of tin using the intracellular dialysis method with potential and ion concentration clamping. After preapplication of 10 microM SnCl2 for 1 min, acetylcholine-induced influx currents decreased by 41%, we compared differences in the effects induced by inorganic and organic tin compounds. Sn(CH3) induced a decrease in the amplitude of acetylcholine-induced currents in the same way as inorganic tin. The effect of Sn(CH3)2 was irreversible and stronger as the preapplication time increased. These results support the previous conclusion that agonist-activated channels are an important target for the actions of toxic metals. It is concluded that direct actions on neuron membranes represent an important component in the modulation of synaptic transmission and that this should be considered in studies of the mechanisms of toxicity of tin.

Hyperforin attenuates various ionic conductance mechanisms in the isolated hippocampal neurons of rat

Effects of hyperforin, an acylphloroglucinol derivative isolated from antidepressive medicinal herb Hypericum perforatum (St. John's Wort), on voltage- and ligand-gated ionic conductances were investigated. Whole-cell patch clamp and concentration clamp techniques on acutely isolated hippocampal pyramidal neurons and on cerebellar Purkinje neurons of rat were used. At concentrations between 3 to 100 μM hyperforin induced a dose and time dependent inward current which completely stabilized within a few seconds. Although 1 μM hyperforin inhibited virtually all investigated conductances (GABA ≥ I Ca(N) > I Na ≥ I Ca(P) ≥ AMPA ≥ I K(A) > NMDA > I K(DR)), its effects on several of them could not be reversed by repeated washings. Dose response studies revealed that although AMPA induced current is inhibited by hyperforin in a competitive manner, these responses are not completely blocked by very high concentration of the agent. On the contrary, however, NMDA receptor-activated ionic conductance could be completely and uncompetitively inhibited by the agent. Taken together these observation not only reconfirm that hyperforin is a major neuroactive component of hypericum extracts but also demonstrate that this structurally unique and naturally abundant molecule is a potent modulation of mechanism involved in the control of neuronal ionic conductances. Various observed effects of hyperforin do not, however, seem to be mediated by one single molecular mechanism of action of the agent.

The mechanism gated by external potassium and sodium controls the resting conductance in hippocampal and cortical neurons

The excitation of densely packed mammalian central neurons is followed by a substantial transitory elevation of external K + concentration. This phenomenon may have a different functional significance depending on how the resting membrane conductance mechanisms react to the changes in the gradient of these ions. We have found that in the hippocampal and cortex neurons of rat a large fraction of the membrane conductance in the vicinity of the resting potential is provided by the K + permeability mechanism which is gated by external K + and Na +. The responses of acutely isolated pyramidal neurons to rapidly altered external [K +] were investigated using the whole-cell patch clamp and concentration clamp techniques. Elevation of [K +] out induced a biphasic inward current at membrane potentials more negative than the reversal potential for K + ions. This current consisted of an “instantaneously” increased leakage component and a slowly activated current (τ=48 ms at 21°C) designated below as I ΔK. The latter demonstrated a first order activation kinetics with a remarkably high Q 10=7.31. I ΔK was absent in the peripheral sensory neurons as well as in the Purkinje neurons. Slow activation of I ΔK was critically dependent on [Na +] out: substitution of the extracellular Na + with choline chloride or Li + led to the “instantaneous” reaction of the membrane to the changes in [K +] out. By slowing down potassium influx, I ΔK may be of importance in preserving densely packed pyramidal neurons from immediate excitation following rapid increases in [K +] out.

Mechanisms of modulation of GABA-induced currents in isolated cerebellar cells by tacrine

Tacrine, when applied by the concentration clamp technique to isolated Purkinje cells from rat cerebellum, dose-dependently reduced the amplitude of GABA-activated chloride currents recorded by the patch clamp technique. Half-maximal inhibition (IC50) of currents activated by 2 μM GABA was observed at a tacrine concentration of 107 μM. Tacrine produced a right shift of the dose-response curves for GABA-induced currents without affecting their peak amplitudes. It is suggested that suppression of GABA-induced currents caused by tacrine can not be attributed to its interaction with the benzodiazepine site of the GABAA receptor.

Control of NO Concentration in Solutions of Nitrosothiol Compounds by Light

We studied the thermal and photolytic decomposition of two S-nitrosothiols, S-nitrosoglutathione (GSNO) and S-nitroso-N-acetylpenicillamine (SNAP), in water or propanol solutions. A "concentration clamp" (relatively constant concentration of NO as a function of time) could be implemented in a closed volume by varying the pH, concentration of nitrovasodilator and intensity of the light source. Depending on the conditions, the light either stimulated NO release or sharply decreased NO concentration in the test solutions. Changes in the absorption spectra of GSNO solutions were monitored as a function of light exposure. Generation of superoxide as a product of a photolytic decomposition reaction of S-nitrosothiols and further oxidation of NO is the most likely mechanism for light suppression of NO concentration.

Analysis of AMPA receptor properties during postnatal development of mouse hippocampal astrocytes.

Glial cells in the mammalian brain express various types of voltage- and ligand-gated ion channels, including glutamate receptors (GluRs) of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-subtype. In the present study we followed developmental changes in the functional properties of AMPA receptor (AMPA-R) channels expressed by astrocytes of the mouse hippocampus between postnatal days (P) 5-35 to learn more about the physiological significance of these glial receptors. A fast concentration clamp technique was applied to cells acutely isolated from the CA1 stratum radiatum subregion to quantitatively analyze rapidly activating and desensitizing receptor responses. The equilibrium responses of glutamate and kainate differed between P5 and P12. Although the maximum current induced by kainate was almost the same at all developmental stages, a steep rise in the maximum glutamate response was observed within the same time range. Between P5 and P12 there was an increase in the potentiation of AMPA-R currents with cyclothiazide (CTZ); at the same time, the dissociation kinetics of CTZ became significantly slower. These changes in the pharmacological properties suggested a variation in splice variant expression. With proceeding maturation, we observed an increase in the degree of desensitization of the glutamate- and AMPA-induced receptor currents. In addition to the shift in flip/flop splicing, these findings could hint at a developmental regulation of RNA editing in the arginine/glycine site. Altogether, the present results demonstrate changes in astrocytic AMPA-R functioning early in postnatal development, although after P12 the receptor properties remained almost constant. Although the overall Ca2+ permeability did not vary during development, the prolonged receptor opening in the early postnatal period causes an enhanced Na+/Ca2+ influx into the immature astrocytes. This could influence glial proliferation and differentiation during CNS ontogenesis.

Opposite effects of interleukin-2 and interleukin-4 on GABA-induced inward currents of dialysed Lymnaea neurons

1. 1. The effects of interleukin-2 (rhIL-2) and interleukin-4 (rhIL-4) were investigated on γ-aminobutyric acid (GABA)-induced inward currents on isolated, identified neurons of Lymnaea stagnalis L. (Mollusca, Gastropoda) by using a concentration clamp technique. 2. 2. It was shown that the interleukins modified the GABA-induced inward current in an opposite direction: rhIL-2 (2–100 U/ml) decreased the peak value of I GABA, in a dose-dependent manner, whereas rhIL-4 (0.2–100 U/ml), on the contrary, potentiated it. Both types of modulation were partially or fully reversible. 3. 3. The reversal potential of I GABA was not shifted by these cytokines. 4. 4. The time-to-peak value and inactivation time constant of the γ-aminobutyric acid (GABA)-induced current was decreased by rhIL-4. The modulatory effect of rhIL-4 was eliminated after conjugation of this cytokine with its antibody. 5. 5. It appears that cytokines could play a role in regulating the neural excitability through GABA-erg mechanisms.

Electrophysiological properties of P2X‐purinoceptors in rat superior cervical, nodose and guinea‐pig coeliac neurones.

1. Whole-cell recordings were made from guinea-pig coeliac, rat nodose and rat superior cervical (SCG) neurones in culture, and currents in response to fast-flow (concentration clamp) application of ATP and other ATP analogues were measured. 2. At a holding potential of -70 mV, ATP evoked inward currents in all neurons. ATP-induced currents reversed at approximately 0 mV and showed strong inward rectification. Halving the external sodium concentration shifted the reversal potential by -15 to -17 mV, while increasing external potassium from 2 to 20 mM produced a 6-10 mV shift in reversal potential. Latency to onset of ATP current was < 1 ms; rise time was concentration dependent with maximum time to peak of 5-20 ms in nodose and coeliac neurones but 80 ms in superior cervical neurones. 3. Threshold concentrations of ATP were 0.1 microM for nodose and coeliac neurones but 10 microM for superior cervical neurones; EC50 values were approximately 3 microM for both nodose and coeliac neurones and 43 microM for superior cervical ganglia. Hill slopes for ATP concentration-response curves were not significantly different from unity in nodose and coeliac neurones whereas the Hill slope in superior cervical neurones was two. 4. 2-MethylthioATP (2-MeSATP) acted as a full agonist in all three neuronal preparations; EC50 values were 0.4, 2.8 and 46 microM for nodose, coeliac and superior cervical neurones, respectively. alpha,beta-Methylene ATP (alpha,beta-MeATP) was a full agonist in nodose and coeliac neurones with EC50 values of 9 and 13 microM, respectively. 5. In superior cervical neurones alpha,beta-MeATP had little or no agonist action but produced a concentration-dependent attenuation of the ATP current. Thus, alpha, beta-MeATP appears to behave as a partial agonist at P2X-purinoceptors in superior cervical neurones. 6. The non-selective purinoceptor antagonists suramin (1-100 microM) and pyridoxal-5'-phosphate (30 microM), as well as the putative P2Y-selective antagonist, Cibacron Blue (30 microM), inhibited all agonist-evoked responses to a similar degree in all three neuronal populations. 7. This study demonstrates that an agonist potency profile of 2-MeSATP > or = ATP > or = alpha,beta-MeATP is characteristic of ligand-gated P2X-purinoceptors in isolated peripheral neurones. We also suggest that the P2X-receptor in superior cervical neurones may represent a distinct subtype of P2X-purinoceptor from that present in nodose and coeliac neurones.

Interaction of concanavalin A and wheat germ agglutinin with Helix acetylcholine receptors

The effects of lectins concanavalin A (Con A) and wheat germ agglutinin (WGA), were studied on acetylcholine (ACh) responses of physically isolated internally dialyzed Helix aspersa neurons using the concentration clamp method and on the binding of [ 3H]α-bungarotoxin to the cluster of neurons. Con A and WGA have different simple sugar specificity and produced different actions on ACh-evoked Cl conductance responses, which were antagonized by Con A (5 μg/ml) but were not altered by WGA. Con A depressed ACh responses when applied extracellularly while it had no effect on ACh responses of the same neuron when added to the intracellular solution, thus indicating that Con A specific glycoproteins are exposed on the surface of the neuron. The studies of the effect of Con A on the properties of the ACh binding site (receptor) have demonstrated, that (1) the onset of desensitization of ACh responses of the dialyzed neurons, determined from the decay of ACh-current from peak to plateau in the continued presence of agonist and best fitted by a double exponential function, was accelerated by Con A; (2) Con A depressed the maximal ACh induced current in a dose response relationship and altered the Hill coefficients; (3) Con A depressed the binding of [ 3H]α-bungarotoxin to the cluster of neurons. These results indicate that Con A receptors on the surface of the neuronal membrane play a regulatory role in the ACh-receptor system and suggest that binding of lectin molecules to their receptors leads to inhibition of binding of ACh to ACh-receptors and to acceleration of the kinetics of desensitization of ACh receptors. All the effects of Con A, that is, on the peak amplitude, desensitization, dose-response relationship of ACh induced current and binding of [ 3H]α-bungarotoxin, could be recovered by D-mannose, a competitive inhibitor of Con A binding to its receptor. The results also demonstrate, that studies of the effects of lectins on dialyzed snail neurons may provide useful information about distribution, properties and functional role of the carbohydrates exposed on the neuronal surface.

The calcium concentration clamp: spikes and reversible pulses using the photolabile chelator DM-nitrophen

New procedures are described for producing brief transients and reversible elevations in [Ca] that can be used to quantitatively control the concentration of cytoplasmic calcium. If the photolabile calcium chelator DM-nitrophen, partially bound to calcium, is exposed to steady illumination, [Ca] can be raised from a few nM to up to 10 μM for durations of 100 ms or longer, depending on light intensity and duration. An association rate of calcium with nitrophen of 1.5 × 10 6 M −1s −1 was estimated from measurements of [Ca] using the fluorescent indicator Fluo-3, and calcium was found to speed the photolysis of nitrophen 2.5-times. Partial photolysis of DM-nitrophen partly loaded with calcium elicits a [Ca] spike of over 100 μM lasting about 1 ms, depending on intensity and duration of the light flash. Simulations of the reactions involved predict changes in Fluo-3 fluorescence measured at high time resolution with a laser scanning confocal microscope. These procedures have been applied in physiological experiments to generate cytoplasmic [Ca] spikes and pulses and study the cellular responses to them.