Rat hippocampal neurons in culture: Ca2+ and Ca2+-dependent K+ conductances

1986 ◽  
Vol 55 (4) ◽  
pp. 751-766 ◽  
Author(s):  
M. Segal ◽  
J. L. Barker
Keyword(s):  
Membrane Potential ◽  
Voltage Clamp ◽  
Hippocampal Neurons ◽  
Outward Current ◽  
Pyramidal Cells ◽  
Equilibrium Potential ◽  
Potential Range ◽  
Current Response ◽  
Experimental Conditions ◽  
Inward Current

Rat hippocampal neurons grown in dissociated cell culture were studied in a medium containing 1 microM tetrodotoxin (TTX) and 25 mM tetraethylammonium (TEA), which eliminated the Na+ and K+ conductances normally activated by depolarizing current injections. In this medium depolarizing current pulses evoked depolarizing regenerative potentials and afterhyperpolarizations in most cells. Both of these events were blocked by close application of Co2+ or Cd2+. These events resemble Ca2+ spikes reported previously in hippocampal pyramidal cells. The membrane potential at which these Ca2+ spikes could be triggered and the rheobase current necessary were dependent on the potential at which the cell was conditioned: the more depolarized the holding potential, the more negative the absolute potential at which a spike could be triggered and the less rheobase current required. The duration of these Ca2+ spikes was also sensitive to the holding potential: the more depolarized the holding level, the longer the duration of the triggered spikes. The amplitude and duration of the Ca2+ spikes were enhanced in a reversible manner by 0.5-1.0 mM 4-aminopyridine (4-AP) delivered in the vicinity of the cell. Two-electrode voltage-clamp analysis of cells studied in TTX, TEA-containing medium revealed an inward current response that peaked in 25-50 ms during depolarizing commands. This response first became detectable during commands to -30 mV. It peaked in amplitude during commands to -10 mV and was enhanced in medium containing elevated [Ca2+]0. It was blocked by either 20 mM Mg2+, 0.2 mM Cd2+, 5 mM Co2+, or 5 mM Mn2+. These results have led us to identify this inward current response as ICa2+. 4-AP enhanced the magnitude and duration of ICa2+ independent of the drug's depressant effects on a transient K+ current also observed under these same experimental conditions. In many but not all cells the Ca2+ spike was followed by a long-lasting hyperpolarization associated with an increase in membrane conductance. This was blocked by Co2+. Under voltage clamp ICa2+ was followed by a slowly developing outward current response that was attenuated by Co2+ or Cd2+. These properties observed under current- and voltage-clamp recording conditions are superficially similar to those previously reported for Ca2+-dependent K+ conductance mechanisms (IC) recorded in these and other membranes. Long-lasting tail currents following activation of IC inverted in the membrane potential range for the K+ equilibrium potential found in these cells.

Rat hippocampal neurons in culture: potassium conductances

1984 ◽  
Vol 51 (6) ◽  
pp. 1409-1433 ◽  
Author(s):  
M. Segal ◽  
J. L. Barker
Keyword(s):  
Time Constant ◽  
Hippocampal Neurons ◽  
Outward Current ◽  
Resting Potential ◽  
Equilibrium Potential ◽  
Transient Outward Current ◽  
Current Response ◽  
Normal Medium ◽  
Inward Current ◽  
Voltage Dependent

Two-electrode voltage-clamp methodology was used to analyze voltage-dependent ionic conductances in 81 rat hippocampal neurons grown in culture for 4-6 wk. Pyramidal and multipolar cells with 15- to 20-micron-diameter cell bodies were impaled with two independent KCl electrodes. The cells had resting potentials of -30 to -60 mV and an average input resistance of about 30 M omega. A depolarizing command applied to a cell maintained in normal medium invariably evoked a fast (2-10 ms) inward current that saturated the current-passing capacity of the system. This was blocked in a reversible manner by application of tetrodotoxin (TTX) (0.1-1.0 microM) near the recorded cell. In the presence of TTX, a depolarizing command evoked a rapidly rising (3-5 ms), rapidly decaying (25 ms) transient outward current reminiscent of "IA" reported in molluscan neurons. This was followed by a more slowly activating (approximately 100 ms) outward current response of greater amplitude that decayed with a time constant of about 2-3 s. These properties resemble those associated with the K+ conductance, IK, underlying delayed rectification described in many excitable membranes. IK was blocked by extracellular application of tetraethylammonium (TEA) but was insensitive to 4-aminopyridine (4-AP) at concentrations that effectively eliminated IA. IA, in turn, was only marginally depressed by TEA. Unlike IK, IA was completely inactivated when the membrane was held at potentials positive to -50 mV. Inactivation was completely removed by conditioning hyperpolarization at -90 mV. A brief hyperpolarizing pulse (10 ms) was sufficient to remove 95% of the inactivation. IA activated on commands to potentials more positive than -50 mV. The inversion potential of the ionic conductance underlying IA and IK was in the range of the K+ equilibrium potential, EK, as measured by the inversion of tail currents; and this potential was shifted in a depolarizing direction by elevated [K+]0. Thus, both current species reflect activation of membrane conductance to K+ ions. Hyperpolarizing commands from resting potentials revealed a time- and voltage-dependent slowly developing inward current in the majority of cells studied. This membrane current was observed in cells exhibiting "anomalous rectification" and was therefore labeled IAR. It was activated at potentials negative to -70 mV with a time constant of 100-200 ms and was not inactivated. A return to resting potential revealed a tail current that disappeared at about EK. IAR was blocked by extracellular CS+ and was enhanced by elevating [K+]0. It thus appears to be carried by inward movement of K+ ions.(ABSTRACT TRUNCATED AT 400 WORDS)

Calcium-dependent current generating the afterhyperpolarization of hippocampal neurons

1986 ◽  
Vol 55 (6) ◽  
pp. 1268-1282 ◽  
Author(s):  
B. Lancaster ◽  
P. R. Adams
Keyword(s):  
Voltage Clamp ◽  
Hippocampal Neurons ◽  
Hippocampal Slices ◽  
Outward Current ◽  
Pyramidal Cells ◽  
Resting Potential ◽  
Tail Current ◽  
Calcium Dependent ◽  
K Current

A single-electrode voltage-clamp technique was employed on in vitro hippocampal slices to examine the membrane current responsible for the slow afterhyperpolarization (AHP) in CA1 pyramidal cells. This was achieved by using conventional procedures to evoke an AHP in current clamp, followed rapidly by a switch into voltage clamp (hybrid clamp). The AHP current showed a dependence on extracellular K+, which was close to that predicted for a K+ current by the Nernst equation. The AHP current could be blocked by Cd2+ or norepinephrine. Although the AHP current showed a requirement for voltage-dependent Ca2+ entry, the current did not show any clear intrinsic voltage dependence. Once activated, AHP current is not turned off by hyperpolarizing the membrane potential. The effects of norepinephrine, Cd2+, and tetraethylammonium (TEA) were used to identify an AHP current component to the outward current evoked by depolarizing voltage commands from holding potentials that approximate to the resting potential for these cells. The AHP current can contribute significantly to the outward current during the depolarizing command. Upon repolarization it is evident as a slow outward tail current. This slow tail current had the same time constant as AHP currents evoked by hybrid clamp. Fast components to the tail currents were also observed. These were sensitive to Cd2+ and TEA. They probably represent a voltage-sensitive gKCa, sometimes termed C-current. The strong sensitivity to voltage and TEA displayed by the conventionally described gKCa (IC) are properties inconsistent with the AHP. It seems likely that the AHP current (IAHP) represents a Ca2+-activated K+ current separate from IC and that these two currents coexist in the same cell.

Cl− Accumulation Does Not Account for the Depolarizing Phase of the Synaptic GABA Response in Hippocampal Pyramidal Cells

1999 ◽  
Vol 82 (2) ◽  
pp. 768-777 ◽  
Author(s):  
Katherine L. Perkins
Keyword(s):  
Guinea Pig ◽  
Short Interval ◽  
Outward Current ◽  
Pyramidal Cells ◽  
Equilibrium Potential ◽  
Inward Current ◽  
Postsynaptic Currents ◽  
Current Curve ◽  
Separate Channel ◽  
Channel Type

It has been proposed that the depolarizing phase of the biphasic synaptic GABA response could be mediated by HCO3 − passing through GABAA channels after dissipation of the transmembrane Cl− gradient due to intracellular Cl− accumulation. To test this hypothesis, giant GABA-mediated postsynaptic currents (GPSCs) were recorded from pyramidal cells in slices of adult guinea pig hippocampus in the presence of 4-aminopyridine. GPSCs consisted of an early outward current (GABAA component) followed by a late inward current (GABAD component). Spontaneous outward inhibitory postsynaptic currents (IPSCs) occurred during the GABADcomponent of the GPSC. GPSCs that were evoked 1–12 s after the preceding GPSC (short interval, siGPSCs) showed no GABADcomponent even though in many cells the amplitude of the siGPSC was greater than the amplitude of the GABAA component of the preceding spontaneous GPSC. In addition, the siGPSC evoked during the GABAD component of a spontaneous GPSC was an outward current. To test whether the siGPSC lacked a GABADcomponent because it was generated predominantly at the soma, where less of an increase in [Cl−]i would occur, picrotoxin was applied to the soma of the pyramidal cell. To the contrary, this focal application of picrotoxin caused less of a reduction in the amplitude of the siGPSC than in the amplitude of the GABAA component of the GPSC. Furthermore when a GPSC and siGPSC were evoked 10 s apart using identical stimuli, the area under the outward current curve was sometimes greater for the siGPSC than for the GPSC, and yet the siGPSC had no inward component. This result indicates that even when the location of Cl− entry was the same, more Cl− could enter the cell during the siGPSC than during the outward component of the GPSC and yet not lead to an inward current. In addition, when the second of two identical stimuli was applied during the inward GABAD component of the first evoked GPSC, the GABAA response it generated was always outward, demonstrating that the equilibrium potential for GABAA responses did not become more positive than the holding potential during a GPSC. Finally, evoking GPSCs at a hyperpolarized potential revealed that the siGPSC actually lacked a GABAD conductance. These results disprove the Cl− accumulation hypothesis of the synaptic depolarizing GABA response and suggest the possibility that a separate channel type may mediate the GABAD component of the GPSC.

Voltage-clamp analysis of the effects of classical conditioning on the hippocampus

1991 ◽  
Vol 65 (4) ◽  
pp. 796-807 ◽  
Author(s):  
J. V. Sanchez-Andres ◽  
D. L. Alkon
Keyword(s):  
Voltage Clamp ◽  
Outward Current ◽  
Pyramidal Cells ◽  
Linear Increase ◽  
Resting Potential ◽  
Equilibrium Potential ◽  
Training Experience ◽  
Ca1 Pyramidal Cells ◽  
Cholinergic Agents ◽  
K Currents

1. Effects of nictitating membrane conditioning on K+ currents of CA1 pyramidal cells of rabbit hippocampus were studied by the use of the single-electrode voltage-clamp (SEVC) technique. 2. IQ, IM, IC, and IAHP were recorded in slices from control animals, showing behavior similar to that previously described for other preparations. IQ developed as an inward current during hyperpolarizing steps to potentials more negative than the K+ equilibrium potential. IM appeared as an inward inactivating relaxation during hyperpolarizing pulses, from potentials slightly more positive than the resting potential (approximately -40 mV). Such depolarization is thought to activate the IM, IC was recorded during long depolarizing pulses as a slow outward current. IAHP appeared during short depolarizing pulses as an outward current peaking at approximately 200 ms after the pulse. Progressively more positive pulses were accompanied by a linear increase of the peak IAHP value. The slope of the IAHP-voltage relation was used for comparison of cells between groups of animals that had different training experience. 3. Responses of control cells to cholinergic agents were similar to those previously characterized in other preparations. Specifically, cholinergic agonists blocked IM and IAHP, partially reduced IC, and did not affect IQ. 4. Conditioning did not affect IQ, IM, and IC but reduced the slope values of the IAHP-voltage relation. This change is consistent with the conditioning-specific afterhyperpolarization (AHP) reduction previously reported. 5. The effect of conditioning on the IAHP but not on the IC, both Ca(2+)-dependent K+ currents, suggests a direct effect on the former, rather than a reduction of ICa2+ or a change in the levels of Cai2+.

Heterogeneous Actions of Serotonin on Interneurons in Rat Visual Cortex

2003 ◽  
Vol 89 (3) ◽  
pp. 1278-1287 ◽  
Author(s):  
Zixiu Xiang ◽  
David A. Prince
Keyword(s):  
Receptor Antagonist ◽  
Receptor Agonist ◽  
Reversal Potential ◽  
Outward Current ◽  
Membrane Conductance ◽  
Pyramidal Cells ◽  
Equilibrium Potential ◽  
Inward Current ◽  
Outward Currents ◽  
Layer V

The effects of serotonin (5-HT) on excitability of two cortical interneuronal subtypes, fast-spiking (FS) and low threshold spike (LTS) cells, and on spontaneous inhibitory postsynaptic currents (sIPSCs) in layer V pyramidal cells were studied in rat visual cortical slices using whole-cell recording techniques. Twenty-two of 28 FS and 26 of 35 LTS interneurons responded to local application of 5-HT. In the group of responsive neurons, 5-HT elicited an inward current in 50% of FS cells and 15% of LTS cells, an outward current was evoked in 41% of FS cells and 81% of LTS cells, and an inward current followed by an outward current in 9% of FS cells and 4% LTS cells. The inward and outward currents were blocked by a 5-HT3 receptor antagonist, tropisetron, and a 5-HT1A receptor antagonist, NAN-190, respectively. The 5-HT–induced inward and outward currents were both associated with an increase in membrane conductance. The estimated reversal potential was more positive than −40 mV for the inward current and close to the calculated K+equilibrium potential for the outward current. The 5-HT application caused an increase, a decrease, or an increase followed by a decrease in the frequency of sIPSCs in pyramidal cells. The 5-HT3 receptor agonist 1-( m-chlorophenyl) biguanide increased the frequency of larger and fast-rising sIPSCs, whereas the 5-HT1Areceptor agonist (±)8-hydroxydipropylaminotetralin hydrobromide elicited opposite effects and decreased the frequency of large events. These data indicate that serotonergic activation imposes complex actions on cortical inhibitory networks, which may lead to changes in cortical information processing.

Ionic basis of the postsynaptic depolarizing GABA response in hippocampal pyramidal cells

1996 ◽  
Vol 76 (6) ◽  
pp. 3886-3894 ◽  
Author(s):  
K. L. Perkins ◽  
R. K. Wong
Keyword(s):  
Hippocampal Slices ◽  
Reversal Potential ◽  
Outward Current ◽  
Pyramidal Cells ◽  
Equilibrium Potential ◽  
Pipette Solution ◽  
Inward Current ◽  
Chloride Accumulation ◽  
Current Sequence ◽  
Ionic Basis

1. Whole cell voltage-clamp recording with recording pipette solutions of differing ionic composition was used to determine the ionic basis of the depolarizing gamma-aminobutyric acid (GABA) response. In the presence of 4-aminopyridine and excitatory amino acid receptor blockers, giant GABA-mediated postsynaptic currents (GPSCs) were recorded from CA3 pyramidal neurons in hippocampal slices from adult guinea pigs. With the GABAB component blocked, the GPSC was composed of an initial outward current (GABAA component) that peaked at 115 ms followed by a late inward current (GABAD component) that peaked at 400-600 ms. 2. Reduction of the intracellular concentration of potassium ([K+]i)resulted in no significant change in the reversal potential of the GABAD component of the GPSC, indicating that it is not a nonspecific cation current. 3. The HCO3- permeability of the channel mediating the GABAD response was assessed by using recording pipette solutions containing three different concentrations of bicarbonate ([HCO3-], 19, 49, and 102 mM). The reversal potential of the GABAD response shifted in the depolarizing direction as the HCO3- equilibrium potential was shifted in the depolarizing direction, indicating that the channel mediating the GABAD response is permeable to HCO3-. The reversal potential of the GABAD response was more sensitive to changes in recording pipette [HCO3-] than the reversal potential of the GABAA response, indicating that the GABAD response is carried by HCO3- to a greater extent than the GABAA response. 4. The outward current-inward current sequence of the biphasic GPSC was reversed to an inward current-outward current sequence by using a high [Cl-]/low [HCO3-] recording pipette solution (40 mM Cl-/6 mM HCO3-), indicating that the GABAA component is more sensitive to changes in [Cl-]i, and the GABAD component is more sensitive to changes in [HCO3-]i. 5. These data indicate that the GABAD component of the GPSC is predominantly carried by HCO3-. While this result supports the recently propsed chloride accumulation model, the model in its present form cannot explain the inward current-outward current polarity sequence of the GPSC recorded with the high [Cl-]/low [HCO3-] intracellular solution. The data obtained using that solution reveal the need for a more expansive chloride accumulation/ depletion model or for a model utilizing two distinct ionotropic GABA channels with different anion permeability ratios to account for the biphasic nature of the GPSC.

scholarly journals Extracellular Hco3− Dependence of Electrogenic Na/Hco3 Cotransporters Cloned from Salamander and Rat Kidney

2000 ◽  
Vol 115 (5) ◽  
pp. 533-546 ◽  
Author(s):  
Irina I. Grichtchenko ◽  
Michael F. Romero ◽  
Walter F. Boron
Keyword(s):  
Membrane Potential ◽  
Voltage Clamp ◽  
Xenopus Oocytes ◽  
Rat Kidney ◽  
Outward Current ◽  
Membrane Vesicles ◽  
Disulfonic Acid ◽  
Ambystoma Tigrinum ◽  
Ph Titration ◽  
Electrode Voltage

We studied the extracellular [HCOabstract 3 −] dependence of two renal clones of the electrogenic Na/HCO3 cotransporter (NBC) heterologously expressed in Xenopus oocytes. We used microelectrodes to measure the change in membrane potential (ΔVm) elicited by the NBC cloned from the kidney of the salamander Ambystoma tigrinum (akNBC) and by the NBC cloned from the kidney of rat (rkNBC). We used a two-electrode voltage clamp to measure the change in current (ΔI) elicited by rkNBC. Briefly exposing an NBC-expressing oocyte to HCOabstract 3 −/CO2 (0.33–99 mM HCOabstract 3−, pHo 7.5) elicited an immediate, DIDS (4,4-diisothiocyanatostilbene-2,2-disulfonic acid)-sensitive and Na+-dependent hyperpolarization (or outward current). In ΔVm experiments, the apparent Km for HCOabstract 3− of akNBC (10.6 mM) and rkNBC (10.8 mM) were similar. However, under voltage-clamp conditions, the apparent Km for HCOabstract 3− of rkNBC was less (6.5 mM). Because it has been reported that SOabstract 3=/HSO abstract 3− stimulates Na/HCO3 cotransport in renal membrane vesicles (a result that supports the existence of a COabstract 3= binding site with which SOabstract 3= interacts), we examined the effect of SOabstract 3=/HSO abstract 3− on rkNBC. In voltage-clamp studies, we found that neither 33 mM SOabstract 4= nor 33 mM SOabstract 3 =/HSOabstract 3− substantially affects the apparent Km for HCO abstract 3−. We also used microelectrodes to monitor intracellular pH (pHi) while exposing rkNBC-expressing oocytes to 3.3 mM HCOabstract 3 −/0.5% CO2. We found that SO abstract 3=/HSOabstract 3 − did not significantly affect the DIDS-sensitive component of the pHi recovery from the initial CO2 -induced acidification. We also monitored the rkNBC current while simultaneously varying [CO2]o, pHo, and [COabstract 3=]o at a fixed [HCOabstract 3−]o of 33 mM. A Michaelis-Menten equation poorly fitted the data expressed as current versus [COabstract 3=]o . However, a pH titration curve nicely fitted the data expressed as current versus pHo. Thus, rkNBC expressed in Xenopus oocytes does not appear to interact with SOabstract 3 =, HSOabstract 3−, or COabstract 3=.

Whole cell current analyses of pancreatic acinar AR42J cells. I. Voltage- and Ca(2+)-activated currents

1991 ◽  
Vol 260 (5) ◽  
pp. C934-C948 ◽  
Author(s):  
K. Kusano ◽  
H. Gainer
Keyword(s):  
Reversal Potential ◽  
Outward Current ◽  
Pancreatic Acinar Cells ◽  
Equilibrium Potential ◽  
Channel Blockers ◽  
Whole Cell ◽  
Inward Current ◽  
Tail Current ◽  
Ar42j Cells ◽  
Conductance Increase

Voltage- and Ca(2+)-activated whole cell currents were studied in AR42J cells, a clonal cell line derived from rat pancreatic acinar cells, using a patch electrode voltage-clamp technique. Four kinds of ionic currents were identified by their ionic dependencies, pharmacological properties, and kinetic parameters: 1) an outward current flow due mainly to a voltage-dependent K(+)-conductance increase, 2) an initial transient inward current due to an Na(+)-conductance increase, 3) transient and long-duration inward current due to a Ca(2+)-conductance increase, and 4) a slowly activating inward current that persists over the duration of the depolarizing pulse and deactivates slowly upon repolarization, producing a slow inward tail current. The slow inward tail current was particularly robust and was interpreted as due to a Ca(2+)-activated Cl(-)-conductance increase, since 1) the generation of this current was blocked by removing the extracellular Ca2+, applying Ca(2+)-channel blockers (Cd2+, nifedipine), or by lowering the intracellular Ca2+ concentration [( Ca2+]i) with EGTA; and 2) the reversal potential (Erev) of the slow inward tail current was close to 0 mV in the control condition (152 mM [Cl-]o/154 mM [Cl-]i), and changes of the [Cl-]o/[Cl )i ratio shifted the Erev toward the predicted Cl- equilibrium potential.

Modulation of Ca2+ and K+ conductances in an identified insect neurone by the activation of an alpha-bungarotoxin-resistant cholinergic receptor

1996 ◽  
Vol 199 (9) ◽  
pp. 1921-1930
Author(s):  
J A David ◽  
R M Pitman
Keyword(s):  
Outward Current ◽  
Cholinergic Receptor ◽  
Equilibrium Potential ◽  
Muscarinic Agonist ◽  
Induced Current ◽  
Current Component ◽  
Experimental Conditions ◽  
Bath Application ◽  
Alpha Bungarotoxin ◽  
Ca2 Channels

The effects of activation of a population of [alpha]-bungarotoxin ([alpha]-bgt)-insensitive cholinergic receptors on the soma of the cockroach fast coxal depressor motor neurone (Df) have been examined under two-electrode voltage-clamp conditions. Activation of these receptors was achieved by bath-application either of acetylcholine (ACh) in the presence of [alpha]-bgt or of the muscarinic agonist McN-A-343 (McN). Since these receptors have been shown previously to respond to some nicotinic agonists, we refer to them as 'McN-sensitive or mixed pharmacological profile muscarinic receptors' (mMAChRs). Activation of these receptors normally results in a biphasic response consisting of an initial outward current component, which reverses near -70 mV, and a later (delayed) inwardly directed current, which is only observed at membrane potentials more positive than -40 to -20 mV. The initial outwardly directed component of the McN-induced current appears to result from an increase in K+ conductance since it reverses at potentials close to the K+ equilibrium potential (EK) (approximately -70 mV under the experimental conditions used) and is blocked by internal Cs+. This increase in K+ conductance is probably due to an increase in Ca2+-activated K+ current (IK,Ca) which is known to form a large proportion of the outward current observed when this neurone is depolarized. The delayed inwardly directed current induced by McN results from suppression of a Ca2+ current (ICa) which, in turn, causes a decrease in IK,Ca. The net effect is a reduction in outward current, because IK,Ca is considerably larger than ICa. Evidence for an action of McN upon Ca2+ channels is provided by experiments in which K+ currents have been suppressed by internal Cs+ to reveal inward currents produced by the movement of Ba2+ through voltage-dependent Ca2+ channels. Ba2+ currents observed under these conditions are suppressed by bath application of McN. The inwardly directed current component of the McN response is unlikely to involve direct regulation of IK,Ca, since McN has no effect upon this current when it is induced by brief intracellular Ca2+ injections. Both the initial outwardly directed component and the delayed inwardly directed component of the McN-induced current were suppressed by intracellular injection of the Ca2+ chelator BAPTA. These observations suggest that a rise in [Ca2+]i mediates the electrophysiological effects of McN in Df somata.

Ca2+-activated Cl− current in cultured myenteric neurons from murine proximal colon

2003 ◽  
Vol 284 (4) ◽  
pp. C839-C847 ◽  
Author(s):  
Sok Han Kang ◽  
Pieter Vanden Berghe ◽  
Terence K. Smith
Keyword(s):  
Membrane Potential ◽  
Channel Blocker ◽  
Reversal Potential ◽  
Outward Current ◽  
Proximal Colon ◽  
Time Dependent ◽  
Equilibrium Potential ◽  
Resting Membrane Potential ◽  
Myenteric Neurons ◽  
Whole Cell Patch Clamp

Whole cell patch-clamp recordings were made from cultured myenteric neurons taken from murine proximal colon. The micropipette contained Cs+ to remove K+ currents. Depolarization elicited a slowly activating time-dependent outward current ( I tdo), whereas repolarization was followed by a slowly deactivating tail current ( I tail). I tdo and I tail were present in ∼70% of neurons. We identified these currents as Cl− currents ( I Cl), because changing the transmembrane Cl− gradient altered the measured reversal potential ( E rev) of both I tdo and I tail with that for I tailshifted close to the calculated Cl− equilibrium potential ( E Cl). I Cl are Ca2+-activated Cl− current [ I Cl(Ca)] because they were Ca2+dependent. E Cl, which was measured from the E rev of I Cl(Ca) using a gramicidin perforated patch, was −33 mV. This value is more positive than the resting membrane potential (−56.3 ± 2.7 mV), suggesting myenteric neurons accumulate intracellular Cl−. ω-Conotoxin GIVA [0.3 μM; N-type Ca2+ channel blocker] and niflumic acid [10 μM; known I Cl(Ca) blocker], decreased the I Cl(Ca). In conclusion, these neurons have I Cl(Ca) that are activated by Ca2+entry through N-type Ca2+ channels. These currents likely regulate postspike frequency adaptation.

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