Distribution and functional properties of 5-HT3 receptors in the rat hippocampal dentate gyrus: a patch-clamp study

1994 ◽  
Vol 71 (5) ◽  
pp. 1935-1947 ◽  
Author(s):  
K. Kawa
Keyword(s):  
Patch Clamp ◽  
Dentate Gyrus ◽  
Granule Cells ◽  
Receptor Subtype ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Inward Current ◽  
Basket Cells ◽  
Inward Currents ◽  
Induced Currents

1. In dentate gyrus of rat hippocampal slices two distinct types of neurons, principal excitatory neurons (granule cells) and local inhibitory neurons (basket cells), could be identified under Nomarski microscopy; I investigated the actions of serotonin using the whole-cell patch-clamp technique. The identification of the neurons was later confirmed by intracellular staining with Lucifer yellow. 2. In both basket cells and granule cells, whole-cell current recordings revealed spontaneous synaptic currents ranging from < 10 pA to > 200 pA in symmetrical Cl- conditions at a holding potential of -63 mV. These currents were blocked by 10 microM bicuculline, indicating that they resulted from the spontaneous activation of GABAergic inputs (which had been morphologically described in both types of neurons). 3. By focal application of serotonin (2–50 microM) to basket cells under current clamp I evoked a train of action potentials superimposed on a baseline membrane depolarization. Under voltage-clamp conditions serotonin evoked an inward current at a holding potential of -63 mV (currents were detectable in approximately 90% of basket cells studied). The inward current was accompanied by a multitude of small inward currents of short duration (< 100 ms) that were found to be due to the stimulation by serotonin of nearby GABAergic presynaptic neurons innervating the recorded neuron. 4. In granule cells (total of 11 cells) serotonin did not produce any responses under conditions similar to those used for basket cells. The occurrence of bicuculline-sensitive spontaneous synaptic current events seemed to increase during the application of serotonin; this phenomenon reflected the excitatory action of serotonin exclusively on GABAergic interneurons. 5. The serotonin-induced inward currents in basket cells were mediated by the 5-HT3 receptor subtype because 1) they were blocked by either metoclopramide (10 microM) or [3-alpha-tropanyl]-1H-indolecarboxylic acid ester (2 nM), the latter being a specific blocker for the 5-HT3 receptor subtype, and 2) almost similar currents were induced by the application of the selective 5-HT3 receptor agonist 2-methyl 5-HT (2–50 microM) or 1-(m-chlorophenyl)-biguanide (0.1–10 microM). 6. Current-voltage (I–V) relations of serotonin-induced currents in basket cells showed that the reversal potential was close to 0 mV in external standard saline and depended on the concentrations of monovalent cations. I–V relations of serotonin-induced currents revealed inward rectification at the membrane potential range of +30 to -60 mV.(ABSTRACT TRUNCATED AT 400 WORDS)

Carbachol induces K+, Cl-, and nonselective cation conductances in T84 cells: a perforated patch-clamp study

1992 ◽  
Vol 263 (4) ◽  
pp. C780-C787 ◽  
Author(s):  
D. C. Devor ◽  
M. E. Duffey
Keyword(s):  
Patch Clamp ◽  
T84 Cells ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Inward Current ◽  
Perforated Patch ◽  
Ionic Conductances ◽  
Inward Currents ◽  
K Current ◽  
Perforated Patch Clamp

We used the perforated patch-clamp technique to examine cell membrane ionic conductances in isolated cells of the human colonic secretory cell line, T84, during exposure to the muscarinic agonist carbachol. Carbachol (100 microM) induced both outward and inward currents when the patch pipette contained a normal intracellular-like solution, the bath contained a normal extracellular-like solution, and the cells were intermittently voltage clamped between K+ and Cl- equilibrium potentials. The outward current was identified as a K+ current that averaged 483 +/- 95 pA, while the inward current averaged 152 +/- 29 pA (n = 15). The outward and inward currents oscillated with a synchronous frequency of 0.036 +/- 0.006 Hz; however, the onset of the K+ current occurred an average of 457 +/- 72 ms before the onset of the inward current. When the pipette contained a high-NaCl solution, the bath contained a Na(+)-gluconate solution, and the cells were intermittently voltage clamped between Cl- and Na+ equilibrium potentials, carbachol induced both Cl- and nonselective cation currents. The Cl- current averaged 455 +/- 73 pA, while the nonselective cation current, averaged 336 +/- 54 pA (n = 14). No difference was observed in the onset of these two currents. These results indicate that carbachol induces three separate ionic conductances in T84 cells. We used the whole cell patch-clamp technique in a previous study of these cells [D. C. Devor, S. M. Simasko, and M. E. Duffey. Am. J. Physiol. 258 (Cell Physiol. 27): C318-C326, 1990] and found that carbachol induced only an oscillating membrane K+ conductance. Thus some unidentified component of the carbachol-sensitive signal transduction pathway is diffusible and may be lost during whole cell patch clamping.

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.

scholarly journals Divalent Cation Block of Inward Currents and Low-Affinity K+ Uptake in Saccharomyces cerevisiae

1999 ◽  
Vol 181 (1) ◽  
pp. 291-297 ◽  
Author(s):  
Stephen K. Roberts ◽  
Marc Fischer ◽  
Graham K. Dixon ◽  
Dale Sanders
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Patch Clamp ◽  
Divalent Cation ◽  
Divalent Cations ◽  
Outward Current ◽  
Alkali Cations ◽  
Patch Clamp Technique ◽  
Inward Current ◽  
Negative Membrane Potential ◽  
Inward Currents

ABSTRACT We have used the patch clamp technique to characterize whole-cell currents in spheroplasts isolated from a trk1Δ trk2Δstrain of Saccharomyces cerevisiae which lacks high- and moderate-affinity K+ uptake capacity. In solutions in which extracellular divalent cation concentrations were 0.1 mM, cells exhibited a large inward current. This current was not the result of increasing leak between the glass pipette and membrane, as there was no effect on the outward current. The inward current comprised both instantaneous and time-dependent components. The magnitude of the inward current increased with increasing extracellular K+and negative membrane potential but was insensitive to extracellular anions. Replacing extracellular K+ with Rb+, Cs+, or Na+ only slightly modulated the magnitude of the inward current, whereas replacement with Li+ reduced the inward current by approximately 50%, and tetraethylammonium (TEA+) and choline were relatively impermeant. The inward current was blocked by extracellular Ca2+ and Mg2+ with apparentKi s (at −140 mV) of 363 ± 78 and 96 ± 14 μM, respectively. Furthermore, decreasing cytosolic K+ increased the magnitude of the inward current independently of the electrochemical driving force for K+influx, consistent with regulation of the inward current by cytosolic K+. Uptake of 86Rb+ by intacttrk1Δ trk2Δ cells was inhibited by extracellular Ca2+ with a Ki within the range observed for the inward current. Furthermore, increasing extracellular Ca2+ from 0.1 to 20 mM significantly inhibited the growth of these cells. These results are consistent with those of the patch clamp experiments in suggesting that low-affinity uptake of alkali cations in yeast is mediated by a transport system sensitive to divalent cations.

scholarly journals Extracellular acidosis activates ASIC-like channels in freshly isolated cerebral artery smooth muscle cells

2010 ◽  
Vol 298 (5) ◽  
pp. C1198-C1208 ◽  
Author(s):  
Wen-Shuo Chung ◽  
Jerry M. Farley ◽  
Alyssa Swenson ◽  
John M. Barnard ◽  
Gina Hamilton ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Cerebral Artery ◽  
Reversal Potential ◽  
Muscle Cells ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Extracellular Acidosis ◽  
Inward Currents ◽  
Induced Currents

Recent studies suggest that certain acid-sensing ion channels (ASIC) are expressed in vascular smooth muscle cells (VSMCs) and are required for VSMC functions. However, electrophysiological evidence of ASIC channels in VSMCs is lacking. The purpose of this study was to test the hypothesis that isolated cerebral artery VSMCs express ASIC-like channels. To address this hypothesis, we used RT-PCR, Western blotting, immunolabeling, and conventional whole cell patch-clamp technique. We found extracellular H+-induced inward currents in 46% of cells tested ( n = 58 of 126 VSMCs, pH 6.5–5.0). The percentage of responsive cells and the current amplitude increased as the external H+ concentration increased (pH6.0, n = 28/65 VSMCs responsive, mean current density = 8.1 ± 1.2 pA/pF). Extracellular acidosis (pH6.0) shifted the whole cell reversal potential toward the Nernst potential of Na+ ( n = 6) and substitution of extracellular Na+ by N-methyl-d-glucamine abolished the inward current ( n = 6), indicating that Na+ is a major charge carrier. The broad-spectrum ASIC blocker amiloride (20 μM) inhibited proton-induced currents to 16.5 ± 8.7% of control ( n = 6, pH6.0). Psalmotoxin 1 (PcTx1), an ASIC1a inhibitor and ASIC1b activator, had mixed effects: PcTx1 either 1) abolished H+-induced currents (11% of VSMCs, 5/45), 2) enhanced or promoted activation of H+-induced currents (76%, 34/45), or 3) failed to promote H+ activation in nonresponsive VSMCs (13%, 6/45). These findings suggest that freshly dissociated cerebral artery VSMCs express ASIC-like channels, which are predominantly formed by ASIC1b.

Temporal Patterns and Depolarizing Actions of Spontaneous GABAA Receptor Activation in Granule Cells of the Early Postnatal Dentate Gyrus

1998 ◽  
Vol 80 (5) ◽  
pp. 2340-2351 ◽  
Author(s):  
Greg S. Hollrigel ◽  
Stephen T. Ross ◽  
Ivan Soltesz
Keyword(s):  
Patch Clamp ◽  
Action Potential ◽  
Dentate Gyrus ◽  
Granule Cell ◽  
Granule Cells ◽  
Temporal Patterns ◽  
Receptor Activation ◽  
Disulfonic Acid ◽  
Whole Cell ◽  
Postsynaptic Currents

Hollrigel, Greg S., Stephen T. Ross, and Ivan Soltesz. Temporal patterns and depolarizing actions of spontaneous GABAA receptor activation in granule cells of the early postnatal dentate gyrus. J. Neurophysiol. 80: 2340–2351, 1998. Whole cell patch-clamp recordings were used to investigate the properties of the γ-aminobutyric acid type A (GABAA) receptor-mediated spontaneous synaptic events in immature granule cells of the developing, early postnatal day (P0–P6) rat dentate gyrus. With Cs-gluconate-filled whole cell patch pipettes at 0 mV in control medium, spontaneous inhibitory postsynaptic currents (sIPSCs) occurred in prominent bursts (peak amplitude of the bursts 406.9 ± 58.4 pA; intraburst IPSC frequency 71.0 ± 12.4 Hz) at 0.05 ± 0.02 Hz in every immature granule cell younger than P7. Between the bursts of IPSCs, lower frequency (1.7 ± 0.7 Hz), interburst IPSCs could be observed. Bicuculline and picrotoxin as well as the intracellularly applied chloride-channel blockers CsF− and 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) abolished the intraburst as well as the interburst IPSCs, indicating that the IPSCs were mediated by GABAA receptor channels. The bursts of IPSCs, but not the interburst IPSCs, were blocked by the simultaneous application of the glutamate receptor antagonists 2-amino-5-phosphovaleric acid and 6-cyano-7-nitroquinoxaline-2,3-dione, indicating the importance of the glutamatergic excitatory drive onto the interneurons in the early postnatal dentate gyrus. The spontaneously occurring excitatory postsynaptic currents in immature granule cells, observable after the intracellular blockade of GABAA receptor channels with CsF− and DIDS, appeared exclusively as single events at low frequencies, i.e., they did not occur in prominent bursts. Gramicidin-based perforated patch-clamp recordings determined that the reversal potential for the burst of IPSCs (−46.6 ± 3.1 mV) was more depolarized than the resting membrane potential (−54.2 ± 4.2 mV) but more hyperpolarized than the action potential threshold (−41.8 ± 1.7 mV). The depolarizing action of the bursts of synaptic events most often evoked only a single action potential per burst. Simultaneous whole cell patch recordings, with KCl-filled patch pipettes at −60 mV in current clamp from pairs of immature granule cells of the developing dentate gyrus, determined that the bursts of IPSPs took place in a similar temporal pattern but with imperfect synchrony in neighboring granule cells (average lag between the onsets of the bursts between granule cell pairs 77.7 ± 8.6 ms). These results show that the spontaneous activation of GABAA receptors in immature dentate granule cells displays unique properties that are distinct from the temporal patterns and biophysical features of spontaneous GABAA receptor activation taking place in the developing Ammon's horn and in the adult dentate gyrus.

CALCIUM CHANNEL CURRENTS IN NEURONES FROM LOCUST (SCHISTOCERCA GREGARIA) THORACIC GANGLIA

1993 ◽  
Vol 177 (1) ◽  
pp. 201-221 ◽  
Author(s):  
H. A. Pearson ◽  
G. Lees ◽  
D. Wray
Keyword(s):  
Calcium Channel ◽  
Single Channel ◽  
Schistocerca Gregaria ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Transient Component ◽  
Thoracic Ganglia ◽  
Steady State Inactivation ◽  
Inward Currents ◽  
Channel Currents

1. Using the patch-clamp technique, Ca2+ channel currents were recorded from neurones freshly isolated from the thoracic ganglia of the desert locust Schistocerca gregaria. 2. In solutions containing 10 mmol l-1 Ba2+ we observed high-voltage-activated whole-cell inward currents with sustained and transient components, both of which had similar steady-state inactivation properties. 3. Substitution of Ca2+ for Ba2+ was found to reduce whole-cell currents, whereas removal of monovalent cations had no effect. 4. Cd2+ (1 mmol l-1) completely blocked the whole-cell current, but at 10 micromolar preferentially inhibited the sustained component without affecting the transient component. 5. Verapamil (1 micromolar) inhibited both current components but appeared to be more selective for the sustained component, whereas nitrendipine (1 micromolar) had no effect on either component. 6. A single-channel recording suggested that the transient component was carried by a low- conductance channel. 7. Certain compounds with insecticidal action (ryanodine, S-bioallethrin, deltamethrin and avermectin) did not affect calcium channel currents in these cells. 8. These data suggest that there are two types of Ca2+ channels present in locust neurones. These channel types have properties differing from the T-, L- and N-type channels found in vertebrates and, furthermore, were not targets for the insecticides we tested.

Characterization of a Hyperpolarization-Activated Inward Current in Cajal-Retzius Cells in Rat Neonatal Neocortex

2000 ◽  
Vol 84 (3) ◽  
pp. 1681-1691 ◽  
Author(s):  
Werner Kilb ◽  
Heiko J. Luhmann
Keyword(s):  
Dissociation Constant ◽  
Reversal Potential ◽  
Hill Coefficient ◽  
Resting Membrane Potential ◽  
Patch Clamp Technique ◽  
Apparent Dissociation Constant ◽  
Membrane Hyperpolarization ◽  
Whole Cell ◽  
Inward Current ◽  
Retzius Cells

Cajal-Retzius cells are among the first neurons appearing during corticogenesis and play an important role in the establishment of cortical lamination. To characterize the hyperpolarization-activated inward current ( I h) and to investigate whether I h contributes to the relatively positive resting membrane potential (RMP) of these cells, we analyzed the properties of I h in visually identified Cajal-Retzius cells in cortical slices from neonatal rats using the whole cell patch-clamp technique. Membrane hyperpolarization to −90 mV activated a prominent inward current that was inhibited by 1 mM Cs+ and was insensitive to 1 mM Ba2+. The activation time constant for I h was strongly voltage dependent. In Na+-free solution, I h was reduced, indicating a contribution of Na+. An analysis of the tail currents revealed a reversal potential of −45.2 mV, corresponding to a permeability coefficient (pNa+/pK+) of 0.13. While an increase in the extracellular K+ concentration ([K+]e) enhances I h, it was reduced by a [K+]e decrease. This [K+]e dependence could not be explained by an effect on the electromotive force on K+ but suggested an additional extracellular binding site for K+ with an apparent dissociation constant of 7.2 mM. Complete Cl−substitution by Br−, I−, or NO3 − had no significant effect on I h, whereas a complete Cl−substitution by the organic compounds methylsulfate, isethionate, or gluconate reduced I h by ∼40%. The I h reduction observed in gluconate could be abolished by the addition of Cl−. The analysis of the [Cl−]e dependence of I h revealed a dissociation constant of 9.8 mM and a Hill-coefficient of 2.5, while the assumption of a gluconate-dependent I h reduction required an unreasonably high Hill-coefficient >20. An internal perfusion with the lidocaine derivative lidocaine N-ethyl bromide blocks I h within 1 min after establishment of the whole cell configuration. An inhibition of I h by 1 mM Cs+ was without an effect on RMP, action potential amplitude, threshold, width, or afterhyperpolarization. We conclude from these results that Cajal-Retzius cells express a prominent I hwith characteristic properties that does not contribute to the RMP.

scholarly journals Effect of Auxin (IAA) on the Fast Vacuolar (FV) Channels in Red Beet (Beta vulgaris L.) Taproot Vacuoles

2020 ◽  
Vol 21 (14) ◽  
pp. 4876
Author(s):  
Zbigniew Burdach ◽  
Agnieszka Siemieniuk ◽  
Waldemar Karcz
Keyword(s):  
Single Channel ◽  
Outward Current ◽  
K Channel ◽  
Single Channels ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Inward Current ◽  
Bath Solution ◽  
Outward Currents ◽  
Inward Currents

In contrast to the well-studied effect of auxin on the plasma membrane K+ channel activity, little is known about the role of this hormone in regulating the vacuolar K+ channels. Here, the patch-clamp technique was used to investigate the effect of auxin (IAA) on the fast-activating vacuolar (FV) channels. It was found that the macroscopic currents displayed instantaneous currents, which at the positive potentials were about three-fold greater compared to the one at the negative potentials. When auxin was added to the bath solution at a final concentration of 1 µM, it increased the outward currents by about 60%, but did not change the inward currents. The imposition of a ten-fold vacuole-to-cytosol KCl gradient stimulated the efflux of K+ from the vacuole into the cytosol and reduced the K+ current in the opposite direction. The addition of IAA to the bath solution with the 10/100 KCl gradient decreased the outward current and increased the inward current. Luminal auxin reduced both the outward and inward current by approximately 25% compared to the control. The single channel recordings demonstrated that cytosolic auxin changed the open probability of the FV channels at the positive voltages to a moderate extent, while it significantly increased the amplitudes of the single channel outward currents and the number of open channels. At the positive voltages, auxin did not change the unitary conductance of the single channels. We suggest that auxin regulates the activity of the fast-activating vacuolar (FV) channels, thereby causing changes of the K+ fluxes across the vacuolar membrane. This mechanism might serve to tightly adjust the volume of the vacuole during plant cell expansion.

Ligand-gated currents of alpha and beta ganglion cells in the cat retinal slice

1994 ◽  
Vol 72 (3) ◽  
pp. 1260-1269 ◽  
Author(s):  
E. D. Cohen ◽  
Z. J. Zhou ◽  
G. L. Fain
Keyword(s):  
Synaptic Transmission ◽  
Beta Cells ◽  
Ganglion Cells ◽  
Excitatory Amino Acid ◽  
Ringer Solution ◽  
Equilibrium Potential ◽  
Patch Clamp Technique ◽  
Pipette Solution ◽  
Inward Current ◽  
Inward Currents

1. We studied the receptor pharmacology of the ligand-gated currents of ON- and OFF- alpha and beta ganglion cells in a cat retinal slice preparation using the whole cell recording variation of the patch-clamp technique. Cat retinal slices were cut in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer and incubated in a bicarbonate-buffered solution. Ganglion cells were voltage clamped at -70 mV in HEPES-buffered Ringer solution. The pipette solution contained a low concentration of Cl- to distinguish mixed cationic from Cl(-)-mediated conductances, and Lucifer yellow (0.5%) was included for identification of the cell type. 2. In Ringer solution containing 1.2 mM Mg2+, current-voltage (I-V) curves of responses to the excitatory amino acid agonist (EAA) N-methyl-D-aspartate (NMDA) (200 microM) revealed a J-shaped function. In Mg(2+)-free Ringer solution containing 200 microM Cd2+ to block synaptic transmission, NMDA (200 microM) elicited an inward current 5-8 times larger at -70 mV. In both conditions I-V curves of the NMDA-induced currents reversed near 0 mV. These results suggest that there are NMDA EAA receptors present directly on the dendrites of alpha and beta ganglion cells. Responses to NMDA were blocked by +/- 2-amino-7-phosphonoheptanoic acid (AP7) (200 microM). 3. In Ringer solution containing 200-1,000 microM Cd2+ to block synaptic transmission, both ON- and OFF- alpha and beta cells responded to kainic acid (10-50 microM), alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA) (20-70 microM), and quisqualic acid (0.1-30 microM) with inward currents that reversed near 0 mV. These responses were blocked by the quinoxaline EAA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (10 microM). The metabotropic agonists 1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) (25 microM) and L-2-amino-4-phosphonobutyric acid (L-APB) (50 microM) and L-2-amino-4-phosphonobutyric acid (L-APB) (50 microM) in the presence of Cd2+ evoked little or no response for all cells tested. 4. In the presence of Cd2+, alpha and beta cells responded to gamma-amino-butyric acid (GABA) (200 microM) and glycine (200 microM) with inward currents that reversed near -35 mV, the calculated chloride equilibrium potential Ecl. Responses to GABA and glycine were both strongly desensitizing. (+)Bicuculline methyl chloride (20 microM) blocked an average of 90% of the inward current evoked by 200 microM GABA on all ganglion cell types.(ABSTRACT TRUNCATED AT 400 WORDS)

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