The mechanism of the releasing action of amphetamine. Uptake, superfusion, and electrophysiological studies on transporter-transfected cells

2000 ◽  
Vol 72 (6) ◽  
pp. 1045-1050 ◽  
Author(s):  
C. Pifl ◽  
H. H Sitte ◽  
H. Reither ◽  
E. A. Singer
Keyword(s):  
Patch Clamp ◽  
Molecular Targets ◽  
Monoamine Transporters ◽  
Transfected Cells ◽  
Whole Cell Patch Clamp ◽  
Sodium Dependent ◽  
Electrophysiological Studies ◽  
Inward Currents ◽  
Vesicular Monoamine Transporters ◽  
The Brain

Amphetamine analogues are able to induce signs of neurotoxicity in the brain. In order to understand this type of neurotoxicity, the interaction of amphetamine with its molecular targets must be elucidated. These molecular targets are plasmalemmal and vesicular monoamine transporters. We investigated the interaction of amphetamine with these transporters in cells transfected with the respective cDNA. Superfusion and whole-cell, patch-clamp experiments were performed, and the toxicity of substrates of the transporters was studied. Amphetamine was taken up by dopamine transporter-expressing cells in a sodium-dependent and cocaine-blockable manner. Furthermore, it elicited inward currents in these cells concentration-dependently. Correlation of uptake, release, and patch-clamp experiments suggest that ion fluxes induced by substrate-gating on transporters may significantly contribute to the releasing action of amphetamine and of other transporter substrates. Dopamine accumulation into serotoninergic terminals depleted of serotonin by 3,4-methylenedioxymethamphetamine was discussed as a mechanism of Ecstasy-toxicity. This is in agreement with a toxic effect of intracellular dopamine which could be demonstrated on our transporter-overexpressing cells. These results, apart from their relevance for the toxicity by amphetamine analogues, may also have bearings on the mechanisms in neurodegenerative diseases affecting monoamine transmitters.

Synaptic Transmission From the Supratrigeminal Region to Jaw-Closing and Jaw-Opening Motoneurons in Developing Rats

2008 ◽  
Vol 100 (4) ◽  
pp. 1885-1896 ◽  
Author(s):  
Shiro Nakamura ◽  
Tomio Inoue ◽  
Kan Nakajima ◽  
Masayuki Moritani ◽  
Kiyomi Nakayama ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Synaptic Transmission ◽  
Motor Nucleus ◽  
Trigeminal Motor Nucleus ◽  
Sensory Inputs ◽  
Optical Responses ◽  
Whole Cell Patch Clamp ◽  
Jaw Opening ◽  
Premotor Neurons ◽  
Inward Currents

The supratrigeminal region (SupV) receives abundant orofacial sensory inputs and descending inputs from the cortical masticatory area and contains premotor neurons that target the trigeminal motor nucleus (MoV). Thus it is possible that the SupV is involved in controlling jaw muscle activity via sensory inputs during mastication. We used voltage-sensitive dye, laser photostimulation, patch-clamp recordings, and intracellular biocytin labeling to investigate synaptic transmission from the SupV to jaw-closing and jaw-opening motoneurons in the MoV in brain stem slice preparations from developing rats. Electrical stimulation of the SupV evoked optical responses in the MoV. An antidromic optical response was evoked in the SupV by MoV stimulation, whereas synaptic transmission was suppressed by substitution of external Ca2+ with Mn2+. Photostimulation of the SupV with caged glutamate evoked rapid inward currents in the trigeminal motoneurons. Gramicidin-perforated and whole cell patch-clamp recordings from masseter motoneurons (MMNs) and digastric motoneurons (DMNs) revealed that glycinergic and GABAergic postsynaptic responses evoked in MMNs and DMNs by SupV stimulation were excitatory in P1–P4 neonatal rats and inhibitory in P9–P12 juvenile rats, whereas glutamatergic postsynaptic responses evoked by SupV stimulation were excitatory in both neonates and juveniles. Furthermore, the axons of biocytin-labeled SupV neurons that were antidromically activated by MoV stimulation terminated in the MoV. Our results suggest that inputs from the SupV excite MMNs and DMNs through activation of glutamate, glycine, and GABAA receptors in neonates, whereas glycinergic and GABAergic inputs from the SupV inhibit MMNs and DMNs in juveniles.

GABA-Mimetic Actions of Withania somnifera on Substantia Gelatinosa Neurons of the Trigeminal Subnucleus Caudalis in Mice

2013 ◽  
Vol 41 (05) ◽  
pp. 1043-1051 ◽  
Author(s):  
Hua Yin ◽  
Dong Hyu Cho ◽  
Soo Joung Park ◽  
Seong Kyu Han
Keyword(s):  
Patch Clamp ◽  
Receptor Antagonist ◽  
Withania Somnifera ◽  
Substantia Gelatinosa ◽  
Patch Clamp Technique ◽  
Pipette Solution ◽  
Whole Cell ◽  
Cell Patch Clamp ◽  
Whole Cell Patch Clamp ◽  
Inward Currents

The plant Withania somnifera (WS), also known as Ashwagandha, has been used widely in traditional medicine systems in India and Nepal (Ayurveda), and has been accepted to cure various ailments. In this study, the whole-cell patch clamp technique was performed to examine the mechanism of action of WS on the SG neurons of the Vc from mouse brainstem slices. In whole-cell patch clamp mode, methanol extract of Withania somnifera (mWS) induced short-lived and repeatable inward currents in all SG neurons tested (31.3±8.51 pA, n = 7) using a high chloride pipette solution. The mWS-induced inward currents were concentration dependent and maintained in the presence of tetrodotoxin (TTX), a voltage gated Na + channel blocker, CNQX, a non-NMDA glutamate receptor antagonist, AP5, an NMDA receptor antagonist and strychnine, a glycine receptor antagonist. The mWS induced currents were blocked by picrotoxin, a GABAA receptor antagonist. These results show that mWS has an inhibitory effects on SG neurons of the Vc through GABAA receptor-mediated activation of chloride ion channels, indicating that mWS contains compounds with sedative effects on the central nervous system. These results also suggest that mWS may be a potential target for modulating orofacial pain processing.

An unusual voltage-gated anion channel found in the cone cells of the chicken retina

1991 ◽  
Vol 6 (1) ◽  
pp. 19-23 ◽  
Author(s):  
Martin Wilson ◽  
Evanna Gleason
Keyword(s):  
Patch Clamp ◽  
Anion Channel ◽  
Chloride Ions ◽  
Patch Clamp Technique ◽  
Adult Chicken ◽  
Voltage Gated ◽  
Cationic Current ◽  
Whole Cell Patch Clamp ◽  
Cone Cells ◽  
Inward Currents

AbstractUsing the whole-cell patch clamp technique, we have examined the voltage-gated currents present in adult chicken cone cells. When calcium and calcium-gated currents are blocked, hyperpolarizing voltage steps elicit slowly increasing inward currents as has been shown for photoreceptors in other species. Unlike the case for other species, chicken cones appear to lack the inward-rectifying cationic current Ih that contributes to the shaping of the photovoltage. Instead of Ih, these cones possess an anionic inward-rectifying current that in kinetics, activation range and probably function is remarkably similar to Ih. This anion channel is unusual in that both nitrate and acetate are more permeant than chloride ions.

scholarly journals K(+) currents in cultured neurones from a polyclad flatworm

2000 ◽  
Vol 203 (20) ◽  
pp. 3189-3198
Author(s):  
S.D. Buckingham ◽  
A.N. Spencer
Keyword(s):  
Steady State ◽  
Patch Clamp ◽  
K Channels ◽  
Type K ◽  
Whole Cell Patch Clamp ◽  
Recovery From Inactivation ◽  
Steady State Inactivation ◽  
The Brain ◽  
K Currents ◽  
Cultured Neurones

Cells from the brain of the polyclad flatworm Notoplana atomata were dispersed and maintained in primary culture for up to 3 weeks. Whole-cell patch-clamp of presumed neurones revealed outwardly directed K(+) currents that comprised, in varying proportions, a rapidly activating (time constant tau =0.94+/−0.79 ms; N=15) and inactivating (tau =26.1+/−1.9 ms; N=22) current and a second current that also activated rapidly (tau =1.1+/−0.2 ms; N=9) (means +/− s.e.m.) but did not inactivate within 100 ms. Both current types activated over similar voltage ranges. Activation and steady-state inactivation overlap and are markedly rightward-shifted compared with most Shaker-like currents (half-activation of 16.9+/−1. 9 mV, N=7, half-inactivation of −35.4+/−3.0 mV, N=5). Recovery from inactivation was rapid (50+/−2.5 ms at −90 mV). Both currents were unaffected by tetraethylammonium (25 mmol l(−1)), whereas 4-aminopyridine (10 mmol l(−1)) selectively blocked the inactivating current. The rapidly inactivating current, like cloned K(+) channels from cnidarians and certain cloned K(+) channels from molluscs and the Kv3 family of vertebrate channels, differed from most A-type K(+) currents reported to date. These findings suggest that K(+) currents in Notoplana atomata play novel roles in shaping excitability properties.

scholarly journals Multi-neuron intracellular recording in vivo via interacting autopatching robots

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Suhasa B Kodandaramaiah ◽  
Francisco J Flores ◽  
Gregory L Holst ◽  
Annabelle C Singer ◽  
Xue Han ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Patch Clamping ◽  
Whole Cell ◽  
Extracellular Recordings ◽  
Mechanical Coupling ◽  
Disease States ◽  
Whole Cell Patch Clamp ◽  
Whole Cell Recordings ◽  
The Brain

The activities of groups of neurons in a circuit or brain region are important for neuronal computations that contribute to behaviors and disease states. Traditional extracellular recordings have been powerful and scalable, but much less is known about the intracellular processes that lead to spiking activity. We present a robotic system, the multipatcher, capable of automatically obtaining blind whole-cell patch clamp recordings from multiple neurons simultaneously. The multipatcher significantly extends automated patch clamping, or 'autopatching’, to guide four interacting electrodes in a coordinated fashion, avoiding mechanical coupling in the brain. We demonstrate its performance in the cortex of anesthetized and awake mice. A multipatcher with four electrodes took an average of 10 min to obtain dual or triple recordings in 29% of trials in anesthetized mice, and in 18% of the trials in awake mice, thus illustrating practical yield and throughput to obtain multiple, simultaneous whole-cell recordings in vivo.

scholarly journals The IGF-derived tripeptide Gly-Pro-Glu is a weak NMDA receptor agonist

2014 ◽  
Vol 112 (6) ◽  
pp. 1241-1245 ◽  
Author(s):  
Christopher E. Vaaga ◽  
Kenneth R. Tovar ◽  
Gary L. Westbrook
Keyword(s):  
Nmda Receptor ◽  
Nmda Receptors ◽  
Glutamate Receptors ◽  
Solution Exchange ◽  
Whole Cell Patch Clamp ◽  
Voltage Dependent ◽  
Inward Currents ◽  
High Concentrations ◽  
High Degree ◽  
The Brain

Glutamate acts as the universal agonist at ionotropic glutamate receptors in part because of its high degree of conformational flexibility. Other amino acids and small peptides, however, can activate N-methyl-d-aspartate (NMDA) receptors, albeit usually with lower affinity and efficacy. Here, we examined the action of glycine-proline-glutamate (GPE), a naturally occurring tripeptide formed in the brain following cleavage of IGF-I. GPE is thought to have biological activity in the brain, but its mechanism of action remains unclear. With its flanking glutamate and glycine residues, GPE could bind to either the agonist or coagonist sites on NMDA receptors, however, this has not been directly tested. Using whole cell patch-clamp recordings in combination with rapid solution exchange, we examined both steady-state currents induced by GPE as well as the effects of GPE on synaptically evoked currents. High concentrations of GPE evoked inward currents, which were blocked either by NMDA receptor competitive antagonists or the voltage-dependent channel blocker Mg2+. GPE also produced a slight attenuation in the NMDA- and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-mediated excitatory postsynaptic currents without altering the paired-pulse ratio. Our results suggest that GPE can activate NMDA receptors but at concentrations well above the expected concentration of GPE in the brain. Therefore, it is unlikely that endogenous GPE interacts with glutamate receptors under normal conditions.

scholarly journals A Novel Membrane Potential-Sensitive Fluorescent Dye Improves Cell-Based Assays for Ion Channels

2002 ◽  
Vol 7 (1) ◽  
pp. 79-85 ◽  
Author(s):  
Deborah F. Baxter ◽  
Martin Kirk ◽  
Amy F. Garcia ◽  
Alejandra Raimondi ◽  
Mats H. Holmqvist ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Ion Channels ◽  
Patch Clamp ◽  
High Throughput Screening ◽  
Fluorescent Dye ◽  
Patch Clamp Recording ◽  
Whole Cell ◽  
Cell Patch Clamp ◽  
Whole Cell Patch Clamp ◽  
Electrophysiological Studies

The study of ion channel-mediated changes in membrane potential using the conventional bisoxonol fluorescent dye DiBAC4(3) has several limitations, including a slow onset of response and multistep preparation, that limit both the fidelity of the results and the throughput of membrane potential assays. Here, we report the characterization of the FLIPR Membrane Potential Assay Kit (FMP) in cells expressing voltage- and ligand-gated ion channels. The steady-state and kinetics fluorescence properties of FMP were compared with those of DiBAC4(3), using both FLIPR and whole-cell patch-clamp recording. Our experiments with the voltage-gated K+ channel, hElk-1, revealed that FMP was 14-fold faster than DiBAC4(3) in response to depolarization. On addition of 60 mM KCl, the kinetics of fluorescence changes of FMP using FLIPR were identical to those observed in the electrophysiological studies using whole-cell current clamp. In addition, KCl concentration-dependent increases in FMP fluorescence correlated with the changes of membrane potential recorded in whole-cell patch clamp. In studies examining vanilloid receptor-1, a ligand-gated nonselective cation channel, FMP was superior to DiBAC4(3) with respect to both kinetics and amplitude of capsaicin-induced fluorescence changes. FMP has also been used to measure the activation of KATP1 and hERG.2 Thus this novel membrane potential dye represents a powerful tool for developing high-throughput screening assays for ion channels.

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