Cultured Insect Mushroom Body Neurons Express Functional Receptors for Acetylcholine, GABA, Glutamate, Octopamine, and Dopamine

1999 ◽  
Vol 81 (1) ◽  
pp. 1-14 ◽  
Author(s):  
M. Cayre ◽  
S. D. Buckingham ◽  
S. Yagodin ◽  
D. B. Sattelle
Keyword(s):  
Patch Clamp ◽  
Mushroom Body ◽  
Gaba Receptor ◽  
Time Course ◽  
Reversal Potential ◽  
Acheta Domesticus ◽  
Free Calcium ◽  
Kenyon Cells ◽  
Inward Currents ◽  
Patch Clamp Electrophysiology

Cayre, M., S. D. Buckingham, S. Yagodin, and D. B. Sattelle. Cultured insect mushroom body neurons express functional receptors for acetylcholine, GABA, glutamate, octopamine, and dopamine. J. Neurophysiol. 81: 1–14, 1999. Fluorescence calcium imaging with fura-2 and whole cell, patch-clamp electrophysiology was applied to cultured Kenyon cells (interneurons) isolated from the mushroom bodies of adult crickets ( Acheta domesticus) to demonstrate the presence of functional neurotransmitter receptors. In all cells investigated, 5 μM acetylcholine (ACh, n = 52) evoked an increase in intracellular free calcium ([Ca2+]i). Similar effects were observed in response to 10 μM nicotine. The ACh response was insensitive to atropine (50 μM) but was reduced by mecamylamine (50 μM) and α-bungarotoxin (α-bgt, 10 μM). ACh-induced inward ion currents ( n = 28, E ACh ∼0 mV) were also blocked by 1 μM mecamylamine and by 1 μM α-bgt. Nicotine-induced inward currents desensitized more rapidly than ACh responses. Thus functional α-bgt–sensitive nicotinic ACh receptors are abundant on all Kenyon cells tested, and their activation leads to an increase in [Ca2+]i. γ-Aminobutyric acid (GABA, 100 μM) triggered a sustained decrease in [Ca2+]i. Similar responses were seen with a GABAA agonist, muscimol (100 μM), and a GABAB agonist, 3-APPA (1 mM), suggesting that more than one type of GABA receptor can affect [Ca2+]i. This action of GABA was not observed when the extracellular KCl concentration was lowered. All cells tested ( n = 26) with patch-clamp electrophysiology showed picrotoxinin (PTX)-sensitive, GABA-induced (30–100 μM) currents with a chloride-sensitive reversal potential. Thus, an ionotropic PTX-sensitive GABA receptor was found on all Kenyon cells tested. Most (61%) of the 54 cells studied responded to l-glutamate (100 μM) application either with a biphasic increase in [Ca2+]i or with a single, delayed, sustained [Ca2+]i increase. Nearly all cells tested (95%, n = 19) responded to (100 μM) l-glutamate with rapidly desensitizing, inward currents that reversed at approximately −30 mV. Dopamine (100 μM) elicited either a rapid or a delayed increase in [Ca2+]i in 63% of the 26 cells tested. The time course of these responses varied greatly among cells. Dopamine failed to elicit currents in patch-clamped cells ( n = 4). A brief decrease in [Ca2+]i was induced by octopamine (100 μM) in ∼54% of the cells tested ( n = 35). However, when extracellular CaCl2 was lowered, octopamine triggered a substantial increase in [Ca2+]i in 35% of the cells tested ( n = 26). No octopamine-elicited currents were detected in patched-clamped cells ( n = 10).

Malate-Regulated Channels Permeable to Anions in Vacuoles of Arabidopsis thaliana

1995 ◽  
Vol 22 (1) ◽  
pp. 115 ◽  
Author(s):  
R Cerana ◽  
L Giromini ◽  
R Colombo
Keyword(s):  
Arabidopsis Thaliana ◽  
Patch Clamp ◽  
High Resistance ◽  
Cultured Cells ◽  
Reversal Potential ◽  
Anion Channels ◽  
Patch Clamp Technique ◽  
Clamp Technique ◽  
Inward Currents ◽  
Cytoplasmic Side

Anion channels in isolated vacuoles of Arabidopsis thaliana cultured cells were studied by means of the patch clamp technique in the whole-vacuole configuration. In symmetrical 100 mM KCl, a high resistance of the membrane at positive potentials inside the vacuole was observed. In symmetrical 100 mM K2-malate positive potentials inside the vacuole elicited slowly developing inward currents, due to the opening of channels, which, according to measurements of reversal potential, are selective for malate. The activation potential of the channels shifted as a function of the cytoplasmic malate concentration, but it was always such that the channels opened only to mediate malate influx into the vacuole. The channels were also permeable to succinate, fumarate and, to a lesser extent, oxaloacetate. In vacuoles preincubated with cytoplas- mic malate, inward currents were also elicited in the presence of KCl or KNO3 at the cytoplasmic side of the tonoplast. Malate channels were different from the cation slow vacuolar-type channels with regard to their sensitivity to changes in the cytoplasmic concentrations of Ca2+ and ATP, and in temperature between 10 and 20�C.

A voltage-gated chloride conductance in rat cultured astrocytes

1986 ◽  
Vol 228 (1252) ◽  
pp. 267-288 ◽  
Keyword(s):  
Time Course ◽  
Reversal Potential ◽  
Outward Current ◽  
Chloride Ions ◽  
Chloride Conductance ◽  
Equilibrium Potential ◽  
Free Calcium ◽  
Patch Clamp Technique ◽  
Bathing Medium ◽  
Cultured Astrocytes

Large voltage-dependent outward currents are recorded with the wholecell patch-clamp technique from rat cultured astrocytes under conditions where an outward movement of potassium ions is excluded (either by blockage of the potassium channels pharmacologically or by replacement of the internal potassium by the impermeant large organic cation N -methyl-( + )-glucamine). The current, which is activated at potentials more positive than —40 to —50 mV, is normally carried by an inward movement of chloride ions. Its reversal potential is the same as the chloride equilibrium potential. With depolarization to +60 mV (for 225 ms) little or no inactivation of the current occurs: with depolarizations to +90 to +110 mV a time-dependent decay is seen. The current, which is often not marked immediately after formation of the whole-cell clamp, generally increases over a period of a few minutes to a maximum (after which it usually declines), as if some as yet unknown intracellular factor keeping the channels closed were being washed away from the membrane. The time course of this phenomenon is not affected by changing of the internal free calcium concentration (from 10 -8 to 10 -6 m) or by an intracellular mixture of cyclic AMP (1 mm), ATP (4 mm) and Mg + (2 mm). The conductance is slightly increased when the chloride of the bathing medium is replaced by bromide; is much reduced on replacement by methylsulphate, sulphate, isethionate, or acetate; and is virtually abolished on replacement by the large anion gluconate. The outward current is inhibited by the disulphonate stilbenes DIDS and SITS; this blocking action was initially partly reversible, although never completely so. It is suggested that the chloride conductance plays a role in the spatial buffering of potassium by astrocytes.

Chloride and cation currents activated by bradykinin in coronary venular endothelial cells

1994 ◽  
Vol 267 (6) ◽  
pp. H2508-H2515
Author(s):  
J. Song ◽  
M. J. Davis
Keyword(s):  
Endothelial Cells ◽  
Time Course ◽  
Reversal Potential ◽  
Disulfonic Acid ◽  
Equilibrium Potential ◽  
K Channel ◽  
Inward Current ◽  
Cation Current ◽  
Inward Currents ◽  
K Current

Bradykinin (BK) is known to activate several types of ion channels in endothelial cells, including a K+ channel and a nonselective cation channel. The predominant BK-activated current in most endothelial cells appears to be an outward, Ca(2+)-activated K+ current. We consistently recorded a rapidly activated, spontaneously inactivated inward current stimulated by BK in bovine coronary venular endothelial cells (CVECs). With the use of a whole cell, perforated patch recording mode, the average magnitude of the current was -293 +/- 38 pA. Simultaneous measurements of current and intracellular Ca2+ concentration ([Ca2+]i) showed that the inward current correlated closely with transient increases in [Ca2+]i due to Ca2+ release from intracellular stores. The current could be blocked by 4,4'-diisothiocyanatostilbene-2, 2'-disulfonic acid (DIDS) but not by La3+, and it persisted in Ca(2+)-free/Na(+)-free solution. When intra- and/or extracellular Cl- concentrations were altered, the reversal potential of the current shifted according to the calculated Cl- -equilibrium potential, indicating that the current was carried primarily by Cl-. Another inward current was also activated by BK. This current was slower to activate, could be blocked by La3+, but was not blocked by DIDS. The time course of the slowly activated current correlated with the plateau phase of the BK-stimulated [Ca2+]i increase, which was similar to the behavior of a nonselective cation current reported previously. We propose that these two currents may contribute to the depolarizations and net inward currents induced by BK in this cell line.

Cl− currents activated via purinergic receptors inXenopus follicles

1998 ◽  
Vol 274 (2) ◽  
pp. C333-C340 ◽  
Author(s):  
Rogelio O. Arellano ◽  
Edith Garay ◽  
Ricardo Miledi
Keyword(s):  
Time Course ◽  
Purinergic Receptors ◽  
Reversal Potential ◽  
Ionic Currents ◽  
Ringer Solution ◽  
Fast Time ◽  
Half Maximal Effective Concentration ◽  
Inward Currents ◽  
Cross Inhibition ◽  
Potency Order

Ionic currents elicited via purinergic receptors located in the membrane of Xenopus follicles were studied using electrophysiological techniques. Follicles responded to ATP-activating inward currents with a fast time course ( F in). In Ringer solution, reversal potential ( E rev) of F in was −22 mV, which did not change with external substitutions of Na+ or K+, whereas solutions containing 50 or 5% of normal Cl−concentration shifted E rev to about +4 and +60 mV, respectively, and decreased F in amplitude, indicating that F in was carried by Cl−. F in had an onset delay of ∼400 ms, measured by application of a brief jet of ATP from a micropipette positioned near the follicle (50 μm). F in was inhibited by 50% in follicles pretreated with pertussis toxin. This suggests a G protein-mediated receptor channel pathway. F in was mimicked by 2-MeSATP and UTP, the potency order (half-maximal effective concentration) was 2-MeSATP (194 nM) > UTP (454 nM) > ATP (1,086 nM). All agonists generated Cl− currents and displayed cross-inhibition on the others. F in activation by acetylcholine also cross-inhibited F in-ATP responses, suggesting that all act on a common channel-activation pathway.

scholarly journals Synthesis and Evaluation of Novel α-Aminoamides Containing Benzoheterocyclic Moiety for the Treatment of Pain

Molecules ◽  
2021 ◽  
Vol 26 (6) ◽  
pp. 1716
Author(s):  
Kun Tong ◽  
Ruotian Zhang ◽  
Fengzhi Ren ◽  
Tao Zhang ◽  
Junlin He ◽  
...  
Keyword(s):  
Ion Channels ◽  
Patch Clamp ◽  
Formalin Test ◽  
Sodium Ion ◽  
Inhibitory Potency ◽  
Voltage Gated ◽  
Patch Clamp Electrophysiology ◽  
Sodium Ion Channels

Novel α-aminoamide derivatives containing different benzoheterocyclics moiety were synthesized and evaluated as voltage-gated sodium ion channels blocks the treatment of pain. Compounds 6a, 6e, and 6f containing the benzofuran group displayed more potent in vivo analgesic activity than ralfinamide in both the formalin test and the writhing assay. Interestingly, they also exhibited potent in vitro anti-Nav1.7 and anti-Nav1.8 activity in the patch-clamp electrophysiology assay. Therefore, compounds 6a, 6e, and 6f, which have inhibitory potency for two pain-related Nav targets, could serve as new leads for the development of analgesic medicines.

scholarly journals Predictive olfactory learning in Drosophila

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Chang Zhao ◽  
Yves F. Widmer ◽  
Sören Diegelmann ◽  
Mihai A. Petrovici ◽  
Simon G. Sprecher ◽  
...  
Keyword(s):  
Synaptic Plasticity ◽  
Dopaminergic Neurons ◽  
Mushroom Body ◽  
Trace Conditioning ◽  
Fruit Fly ◽  
Olfactory Learning ◽  
Shock Strength ◽  
Behavioral Experiments ◽  
Kenyon Cells ◽  
Output Neurons

AbstractOlfactory learning and conditioning in the fruit fly is typically modelled by correlation-based associative synaptic plasticity. It was shown that the conditioning of an odor-evoked response by a shock depends on the connections from Kenyon cells (KC) to mushroom body output neurons (MBONs). Although on the behavioral level conditioning is recognized to be predictive, it remains unclear how MBONs form predictions of aversive or appetitive values (valences) of odors on the circuit level. We present behavioral experiments that are not well explained by associative plasticity between conditioned and unconditioned stimuli, and we suggest two alternative models for how predictions can be formed. In error-driven predictive plasticity, dopaminergic neurons (DANs) represent the error between the predictive odor value and the shock strength. In target-driven predictive plasticity, the DANs represent the target for the predictive MBON activity. Predictive plasticity in KC-to-MBON synapses can also explain trace-conditioning, the valence-dependent sign switch in plasticity, and the observed novelty-familiarity representation. The model offers a framework to dissect MBON circuits and interpret DAN activity during olfactory learning.

Paired-pulse facilitation in the dentate gyrus: a patch-clamp study in rat hippocampus in vitro

1994 ◽  
Vol 72 (1) ◽  
pp. 326-336 ◽  
Author(s):  
M. Andreasen ◽  
J. J. Hablitz
Keyword(s):  
Patch Clamp ◽  
Dentate Gyrus ◽  
Time Constant ◽  
Rise Time ◽  
Time Course ◽  
Nmda Antagonist ◽  
Paired Pulse ◽  
Stimulation Intensity ◽  
Paired Pulse Facilitation ◽  
Epsc Amplitude

1. Whole-cell patch-clamp recordings were used to study paired-pulse facilitation (PPF) of the lateral perforant path input to the dentate gyrus in thin hippocampal slices. 2. Orthodromic stimulation of the lateral perforant pathway evoked a excitatory postsynaptic current (EPSC) with a latency of 3.3 +/- 0.1 ms (mean +/- SE) that fluctuated in amplitude. The EPSC had a rise time (10-90%) of 2.79 +/- 0.06 ms (n = 35) and decayed with a single exponential time course with a time-constant of 9.14 +/- 0.24 ms (n = 35). No correlation was found between the amplitude of the EPSC and the rise time or decay time-constant. The non-N-methyl-D-aspartate (NMDA) antagonist 6-cyano-7-nitroquinoxaline-2,3-dione completely blocked the EPSC whereas the NMDA antagonist D-aminophosphonovaleric acid (APV) had modest effects. 3. When a test (T-)EPSC was preceded at an interval of 100 ms by a conditioning (C-)EPSC, a significant increase in the amplitude of the T-EPSC was seen in 38 out of 44 trials analyzed from a total of 27 granule cells. The average amount of PPF was 35.7 +/- 2.1%. There was no apparent correlation between the amount of PPF and the stimulation intensity or mean amplitude of the C-EPSC. The time course of the facilitated T-EPSC was not significantly different from that of the C-EPSC. 4. No correlation was found between the amplitude of the C-EPSC and that of the T-EPSC. Estimates of quantal content (mcv) were determined by calculating the ratio of the squared averaged EPSC amplitude (from 48 responses) to the variance of these responses (M2/sigma 2) whereas quantal amplitudes (qcv) were estimated by calculating the ratio of the response variance to average EPSC amplitude (sigma 2/M). PPF was found to be associated with an average increase in mcv of 64.8 +/- 7.2% (n = 38) whereas qcv was decreased by 12.1 +/- 3.8%. 5. The time course of PPF was studied by varying the interval between the C- and T-pulse from 10 to 400 ms while keeping the stimulation intensity constant. Maximal facilitation of the T-EPSC was obtained with interpulse intervals < or = 25 ms where the average facilitation amounted to approximately 70% (n = 6). The decline of facilitation was nearly exponential and was no longer evident with intervals > 350 ms.(ABSTRACT TRUNCATED AT 400 WORDS

scholarly journals Isolating Nuclei from Cultured Cells for Patch-Clamp Electrophysiology of Intracellular Ca 2+ Channels

2013 ◽  
Vol 2013 (9) ◽  
pp. pdb.prot073056 ◽  
Author(s):  
Don-On Daniel Mak ◽  
Horia Vais ◽  
King-Ho Cheung ◽  
J. Kevin Foskett
Keyword(s):  
Patch Clamp ◽  
Cultured Cells ◽  
Intracellular Ca ◽  
Patch Clamp Electrophysiology

Permeation and gating properties of a cloned renal K+ channel

1995 ◽  
Vol 268 (2) ◽  
pp. C389-C401 ◽  
Author(s):  
S. Chepilko ◽  
H. Zhou ◽  
H. Sackin ◽  
L. G. Palmer
Keyword(s):  
Single Channel ◽  
Reversal Potential ◽  
Cation Binding ◽  
K Channel ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Membrane Hyperpolarization ◽  
Channel Current ◽  
Inward Currents ◽  
Kinetics Of

The renal K+ channel (ROMK2) was expressed in Xenopus oocytes, and the patch-clamp technique was used to assess its conducting and gating properties. In cell-attached patches with 110 mM K+ in the bath and pipette, the reversal potential was near zero and the inward conductance (36 pS) was larger than the outward conductance (17 pS). In excised inside-out patches the channels showed rectification in the presence of 5 mM Mg2+ on the cytoplasmic side but not in Mg(2+)-free solution. Inward currents were also observed when K+ was replaced in the pipette by Rb+, NH4+, or thallium (Tl+). The reversal potentials under these conditions yielded a selectivity sequence of Tl+ > K+ > Rb+ > NH4+. On the other hand, the slope conductances for inward current gave a selectivity sequence of K+ = NH4+ > Tl+ > Rb+. The differences in the two sequences can be explained by the presence of cation binding sites within the channel, which interact with Rb+ and Tl+ more strongly and with NH4+ less strongly than with K+. Two other ions, Ba2+ and Cs+, blocked the channel from the outside. The effect of Ba2+ (1 mM) was to reduce the open probability of the channels, whereas Cs+ (10 mM) reduced the apparent single-channel current. The effects of both blockers are enhanced by membrane hyperpolarization. The kinetics of the channel were also studied in cell-attached patches. With K+ in the pipette the distribution of open times could be described by a single exponential (tau 0 = 25 ms), whereas two exponentials (tau 1 = 1 ms, tau 2 = 30 ms) were required to describe the closed-time distribution. Hyperpolarization of the oocyte membrane decreased the open probability and tau 0, and increased tau 1, tau 2, and the number of long closures. The presence of Tl+ in the pipette significantly altered the kinetics, reducing tau 0 and eliminating the long-lived closures. These results suggest that the gating of the channel may depend on the nature of the ion in the pore.

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