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PubMed1996

Human alpha4beta2 neuronal nicotinic acetylcholine receptor in HEK 293 cells: A patch-clamp study.

The cloning and expression of genes encoding for the human neuronal nicotinic acetylcholine receptors (nAChRs) has opened new possibilities for investigating their physiological and pharmacological properties. Cells (HEK 293) stably transfected with two of the major brain subunits, alpha4 and beta2, were characterized electrophysiologically using the patch-clamp technique. Fast application of the natural ligand ACh can evoke currents up to 3500 pA, with an apparent affinity (EC50) of 3 microM and a Hill coefficient of 1.2. The rank order of potency of four nAChR ligands to activate human alpha4beta2 receptors is (-)-nicotine > ACh > (-)-cytisine > ABT-418. At saturating concentrations, the efficacy of these ligands is ABT-418 >> (-)-nicotine > ACh >> (-)-cytisine > GTS-21 (previously named DMXB). Coapplication of 1 microM ACh with known nAChR inhibitors such as dihydro-beta-erythroidine and methyllycaconitine reversibly reduces the current evoked by the agonist with respective IC50 values of 80 nM and 1.5 microM. The current-voltage relationship of human alpha4beta2 displays a strong rectification at positive potentials. Experiments of ionic substitutions suggest that human alpha4beta2 nAChRs are permeable to sodium and potassium ions. In the "outside-out" configuration, ACh evokes unitary currents (main conductance 46 pS) characterized by a very fast rundown. Potentiation of the ACh-evoked currents is observed when the extracellular calcium concentration is increased from 0.2 to 2 mM. In contrast, however, a reduction of the evoked currents is observed when calcium concentration is elevated above 2 mM.

Calcium↗
PubMed1998

Scanning electron microscopy and gramicidin patch clamp recordings of microvillous receptor neurons dissociated from the rat vomeronasal organ.

Vomeronasal organs from female rats were dissociated and isolated microvillous receptor neurons were studied. The isolated receptor neurons kept the typical bipolar shape which they have in situ as observed by scanning electron microscopy. We applied the perforated patch-clamp technique using the cation-selective ionophore gramicidin on freshly isolated and well differentiated receptor neurons. The mean resting potential was -58+/-14 mV (n=39). The contribution of the sodium pump current to the resting potential was demonstrated by lowering the K+ concentration in the bath or by application of 100 microM dihydro-ouabain. The input resistance was in the range of 1-6 GOmega and depolarizing current pulses of a few pA were sufficient to trigger overshooting action potentials. In voltage clamp conditions a fast transient sodium inward current and a sustained outward potassium current were activated by membrane depolarization. These observations indicate that freshly isolated vomeronasal receptor neurons of rats can be recorded, using gramicidin, with little modification of the intracellular content. Their electrophysiological properties are very similar to those observed in situ. Four out of eight female vomeronasal receptor cells were depolarized by diluted rat male urine.

Animals↗
PubMed1996

Patch-clamping arthropod olfactory receptor neurons to study mechanisms of olfactory transduction.

The olfactory organ of arthropods such as lobsters and insects consists of an array of hair-like sensilla located on the antenna. Each sensillum contains from two to several hundred primary olfactory receptor neurons. The receptor neurons can be patch-clamped in three different types of preparations: intact cells in situ, cultured cells and outer dendrites. These preparations permit using a wide range of experimental strategies to study mechanisms of olfactory transduction. The ability to integrate data from three complementary preparations is a particular advantage of using arthropod models to understand how odor information is encoded by the primary receptor cell in olfaction.

Animals↗
PubMed1988

Patch clamp analysis of Na channel gating in mammalian myocardium: reconstruction of double pulse inactivation and voltage dependence of Na currents.

Isolated ventricular cells of the mouse heart were prepared by an enzyme digestion procedure. Unitary Na currents were recorded with the patch clamp technique from cell attached patched. Macroscopic Na currents were obtained as mean of 38 consecutive sweeps of cell attached patches with up to 100 channels each. Double pulse inactivation of macroscopic currents showed an increase of the test current amplitude at test pulse potential Vt = -30 mV after short prepulses at prepulse potential Vp = -50 mV. The open time distribution of single channels could be fitted monoexponentially yielding a mean open time tau 0. Between -70 mV and -20 mV tau 0 showed a bell shaped voltage dependence. The probability to record an empty sweep PA (0) had a minimum value at -50 mV and increased towards less negative potentials. The current-voltage relation of the unitary current was linear between -60 mV and -20 mV yielding a slope conductance of 18.5 pS at room temperature. There was no indication of the existence of more than a single unitary current level. For the apparent peak open probability of a Na channel a sigmoidal voltage dependence was found between -70 mV and -20 mV. Single channel recording reveals that the time course of double pulse inactivation coincides with the increase in the number of empty sweeps leaving tau 0 unchanged. The Markov models with two closed states, one open and one inactivated, were used for quantitative analysis. Useful were only models allowing inactivation both from at least one of the closed states and from the open state. For both model types a set of rate constants was calculated from single channel data and from the time course of the opening probability at -50 mV and -30 mV, respectively. The model with the allowed inactivation from the second closed state (M1) was superior to that with the allowed inactivation from the first closed state (M3) by the prediction of the time course of the early double pulse inactivation at Vp = 50 mV and Vt = 30 mV and, based on these data, by the prediction of the amplitude of Na currents at -40 mV and -60 mV.

Animals↗
PubMed1987

Patch-clamp studies of isolated mouse olfactory receptor neurons.

Olfactory receptor neurons isolated from embryonic, neonatal, and adult mice were studied using the patch-clamp technique. Several distinct types of ion channels were characterized in patches of membrane from the neuronal soma and the dendritic knob of receptor neurons, including a 130-pS Ca++-activated K+ channel with voltage-dependent kinetics, an 80-pS Ca++-activated K+ channel with voltage-insensitive kinetics, a 25-pS K+ channel with properties similar to inward rectifiers, and a 40-pS K+ channel that was activated and then inactivated by rapid depolarization. Evidence of large-conductance (greater than 200 pS) Cl- channels, which were Ca++ insensitive and increasingly active at depolarizing membrane potentials, and voltage-activated Ca++ channels (16 pS) was also obtained. From K+ channel activity recorded from cell-attached patches, the intracellular [Ca++] was inferred to be below 0.1 microM, and the membrane potential was inferred to be approximately -50 mV. The receptor neurons had high input resistances, and action potentials could be elicited by picoampere amounts of depolarizing current. The receptor neurons responded to applied odorant molecules and to forskolin with increases in membrane conductance. These results provide a description of the membrane properties of olfactory receptor neurons and a basis for understanding their electrical activity and response to odorants.

Animals↗
PubMed1998

Patch-clamp and amperometric recordings from norepinephrine transporters: channel activity and voltage-dependent uptake.

Transporters for the biogenic amines dopamine, norepinephrine, epinephrine and serotonin are largely responsible for transmitter inactivation after release. They also serve as high-affinity targets for a number of clinically relevant psychoactive agents, including antidepressants, cocaine, and amphetamines. Despite their prominent role in neurotransmitter inactivation and drug responses, we lack a clear understanding of the permeation pathway or regulation mechanisms at the single transporter level. The resolution of radiotracer-based flux techniques limits the opportunities to dissect these problems. Here we combine patch-clamp recording techniques with microamperometry to record the transporter-mediated flux of norepinephrine across isolated membrane patches. These data reveal voltage-dependent norepinephrine flux that correlates temporally with antidepressant-sensitive transporter currents in the same patch. Furthermore, we resolve unitary flux events linked with bursts of transporter channel openings. These findings indicate that norepinephrine transporters are capable of transporting neurotransmitter across the membrane in discrete shots containing hundreds of molecules. Amperometry is used widely to study neurotransmitter distribution and kinetics in the nervous system and to detect transmitter release during vesicular exocytosis. Of interest regarding the present application is the use of amperometry on inside-out patches with synchronous recording of flux and current. Thus, our results further demonstrate a powerful method to assess transporter function and regulation.

Carrier Proteins↗
PubMed2000

Patch-clamp study of neurons and glial cells in isolated myenteric ganglia.

Most of the physiological information on the enteric nervous system has been obtained from studies on preparations of the myenteric ganglia attached to the longitudinal muscle layer. This preparation has a number of disadvantages, e.g., the inability to make patch-clamp recordings and the occurrence of muscle movements. To overcome these limitations we used isolated myenteric ganglia from the guinea pig small intestine. In this preparation movement was eliminated because muscle was completely absent, gigaseals were obtained, and whole cell recordings were made from neurons and glial cells. The morphological identity of cells was verified by injecting a fluorescent dye by micropipette. Neurons displayed voltage-gated inactivating inward Na(+) and Ca(2+) currents as well as delayed-rectifier K(+) currents. Immunohistochemical staining confirmed that most neurons have Na(+) channels. Neurons responded to GABA, indicating that membrane receptors were retained. Glial cells displayed hyperpolarization-induced K(+) inward currents and depolarization-induced K(+) outward currents. Glia showed large "passive" currents that were suppressed by octanol, consistent with coupling by gap junctions among these cells. These results demonstrate the advantages of isolated ganglia for studying myenteric neurons and glial cells.

Animals↗
PubMed1993

Double whole-cell patch-clamp characterization of gap junctional channels in isolated insect epidermal cell pairs.

Double whole-cell patch-clamp methods were used to characterize junctional membrane conductances in epidermal cell pairs isolated from the prepupal integument of the flour beetle, Tenebrio molitor. The mean initial junctional conductance in 267 cell pairs was 9.5 +/- 1.0 nS (range 0-95 nS). Well-coupled cell pairs uncoupled spontaneously with a half-time of 7.6 min. Adding 5 mM ATP to the pipette solution stabilized coupling with less than a 50% drop occurring after 30 min. Nonjunctional membrane potential was the major determinant of junctional conductance with transjunctional potential playing a minor role. Junctional conductance approached 0 pA at nonjunctional membrane potentials greater than 0 mV and increased with hyperpolarization. The voltage at half-maximal conductance was -26 mV. The time course of the reversible changes in junctional conductance were slow (< or = 30 sec) with time-dependent decay occurring faster and recovery occurring slower with increasing depolarization. Single gap junctional channel activity was recorded in uncoupling cell pairs and in poorly coupled ATP-stabilized cell pairs. One main single channel conductance was observed in each cell pair. The mean single channel conductances from all cell pairs in this study ranged from 197-347 pS (mean 248 pS). Single channel conductance was linear over the +/- 60 mV transjunctional voltage range tested. A broad range of subconductance states of the main state representing 5% of the total open time of measurable main state events was observed. Single channel activity was strongly dependent on the nonjunctional membrane potential, increasing with hyperpolarization.

Adenosine Triphosphate↗
PubMed1990

A novel type of nicotinic receptor in the rat central nervous system characterized by patch-clamp techniques.

We present a functional characterization of a neuronal nicotinic receptor in the CNS using patch-clamp techniques and a preparation of acutely isolated neurons from the medial habenular nucleus of 10- to 20-d-old rats. The salient pharmacological and electrophysiological properties of this nicotinic response are (1) its association with a channel that is relatively nonselective for cations and has a unitary conductance of 26.2 (+)+/- 5pS at room temperature; (2) its insensitivity to alpha-bungarotoxin and to neuronal bungarotoxin; (3) its activation by the ganglionic nicotinic agonists nicotine, 1,1-dimethyl-4-phenylpiperazinium and cytisine and its blocking by several nicotinic antagonists, mecamylamine, hexamethonium, d-tubocurarine, and dihydro-beta-erythroidine. The combination of these properties has not been reported for any other known type of nicotinic receptor.

Acetylcholine↗
PubMed1998

Patch-clamp recordings of membrane currents evoked during natural synaptic activity in sympathetic neurons.

Membrane currents elicited by colonic distension and by electrical stimulation of the intermesenteric nerve containing colonic afferent nerve fibres were recorded from neurons of the mouse superior mesenteric ganglion at 20 degrees C with the whole-cell patch-clamp method. Electrically-evoked excitatory postsynaptic currents reversed at -3.5 mV. At membrane holding voltages of -70 mV and -110 mV, the excitatory postsynaptic currents were characterized by a single exponential decay with a mean (+/- S.E.M.) time-constant of 17.5 +/- 1.3 ms and 15.5 +/- 2.3 ms, respectively. Colonic distension evoked a series of the excitatory postsynaptic currents which ranged in amplitude from 10 to 700 pA (at a membrane holding voltage of -70 mV). Hexamethonium (100 microM) applied only to the ganglion abolished both electrically- and distension-evoked excitatory postsynaptic currents, suggesting activation of nicotinic acetylcholine receptors. The decay time-course of distension-evoked single excitatory postsynaptic currents was characterized by one, or, less commonly, by two exponentials. The decay time-constant histograms of distension-evoked single excitatory postsynaptic currents exhibited main kinetic components of 8.1 +/- 2.3 ms and 8.2 +/- 2.5 ms (peak +/- S.D.) at -70 and -110 mV membrane holding voltages, respectively. Longer time-constants ranging up to 51 ms were also observed. The number of the distension-evoked excitatory postsynaptic currents with a decay time-constant higher than 20 ms, as well as their mean amplitude, were significantly lower at -110 mV than at -70 mV membrane potential levels, in contrast to the currents with a decay time-constant lower than or equal to 20 ms. The results suggest that colonic afferent nerve fibres activate in the mouse superior mesenteric ganglion neurons a few populations of the postsynaptic nicotinic acetylcholine receptors with different channel kinetics, which are characterized by a lack of voltage sensitivity within -70 to -110 mV membrane potential range, except those with comparatively slow channel kinetics, which are possibly blocked by membrane hyperpolarization.

Animals↗
PubMed1994

A patch-clamp study on a novel gamma-aminobutyric acid activated chloride channel of crayfish deep extensor abdominal muscle.

The patch-clamp technique was used to study the effect of rapid pulses of gamma-aminobutyric acid (GABA) on excised membrane-patches in the outside-out configuration of the deep extensor abdominal muscle (DEAM) of crayfish. Channel currents reversed at the equilibrium potential of Cl- and were blocked by picrotoxin. Rare channel openings were elicited by 0.1 mM GABA, and a saturating open probability of 0.9 was reached with 10 nM GABA. The investigated channel was only sensitive to GABA and is different from a previously described GABAergic channel in crayfish that is in addition sensitive to acetylcholine and glutamate and shows three subconductance states.

Abdominal Muscles↗
PubMed2006

Patch clamp reveals powerful blockade of the mitochondrial permeability transition pore by the D2-receptor agonist pramipexole.

The dopamine-D2-agonist pramipexole (PPX) was tested for blocking mitochondrial permeability transition (PT) in order to give a possible explanation for its neuroprotective effect seen in PPX-treated Parkinson's disease patients. Patch-clamp techniques for studying single-channel currents in the inner mitochondrial membrane and large-amplitude swelling of energized mitochondria were used to study PPX action on the permeability transition pore (PTP), a key player in the mitochondrial route of the apoptotic cascade. Identity of the PTP was proven by measuring the concentration-response relation for cyclosporin A-blockade (IC50=26 nM). PPX inhibits the PTP reversibly with an IC50 of 500 nM, which is close to the values determined earlier as plasma concentrations after PPX medication in patients. Interaction of PPX with the PTP is further supported by demonstrating that it abolished Ca2+-triggered swelling in functionally intact mitochondria. Blockade of the PTP by PPX was attenuated by increasing concentrations of inorganic phosphate and by acidification. We suggest that PPX could exert part of its neuroprotective effect by inhibition of the PTP and thus, probably, blocking of the mitochondrial pathway of the apoptosis cascade.

Animals↗
PubMed1994

Gramicidin perforated patch-clamp technique reveals glycine-gated outward chloride current in dissociated nucleus solitarii neurons of the rat.

1. The inhibitory response of exogenously applied glycine was investigated in freshly dissociated rat nucleus tractus solitarii neurons under whole cell configuration using new perforated patch-clamp technique termed "gramicidin perforated patch technique," which maintains intact intracellular Cl- concentrations. 2. Using the gramicidin perforated patch technique, at a holding potential (VH) of -45 mV, glycine induced outward currents in a concentration-dependent manner with a EC50 of 4.0 x 10(-5) M and at a Hill coefficient of 1.5. In contrast, using the nystatin perforated patch technique, glycine induced inward currents at the same VH in a concentration-dependent manner with an EC50 of 4.9 x 10(-5) M and at a Hill coefficient of 1.2. 3. The glycine-induced outward currents were blocked by strychnine in a concentration dependent manner with an IC50 of 2.2 x 10(-8) M. The blockade was competitive. 4. The current-voltage relationship for the 10(-5) M glycine response showed a clear outward rectification. 5. Ten-fold change of extracellular Cl- with a large impermeable anion resulted in a 65 mV shift of the reversal potential of glycine-induced currents (EGly), indicating that the membrane behaves like a Cl- electrode in the presence of glycine. 6. The intracellular Cl- activity calculated from the EGly ranged from 7.3 to 18.2 mM, with a mean value of 13.3 mM. 7. The values of EGly in the individual neurons were significantly negative to the resting membrane potentials, suggesting the existence of active transport of Cl-.

Animals↗
PubMed2006

From Galvani to patch clamp: the development of electrophysiology.

The development of electrophysiology is traced from the early beginnings represented by the work of the Dutch microscopist, Jan Swammerdam, in the 17th century through the first notion of an aqueous transmembrane pore as a substrate of excitability made by Luigi Galvani in late 18th century to the invention late in the 20th century of the patch-clamp technique by Erwin Neher and Bert Sakmann.

Animals↗
PubMedDate not supplied

Patch-clamp recording of human retinal photoreceptors and bipolar cells.

Photoreceptors and retinal bipolar cells are considered as nonspiking neurons; however, we recently showed that human rod photoreceptors can generate sodium action potentials in response to membrane depolarization from membrane potentials of -60 or -70 mV (Kawai et al., Neuron 30 [2001] 451). We performed patch-clamp recording of human cone photoreceptors and retinal bipolar cells to examine whether functional voltage-gated sodium channels are expressed in these cells as well as rod photoreceptors. Under current-clamp conditions, the injection of depolarizing current steps into a cone photoreceptor-induced marked action potentials. These action potentials were blocked by 1 microM tetrodotoxin, a voltage-gated sodium channel blocker. Under voltage-clamp conditions, depolarizing voltage steps-induced a fast transient inward current in several bipolar cells (n = 4/78). This current was activated from -70 to + 20 mV (maximal at -10 mV) and inactivated within 5 ms. The 10-90% rise time of this current was shorter than another inward current (less than one-hundredth). These results indicate that human cones and bipolar cells express voltage-gated sodium channels as rod photoreceptors. Sodium channels may serve to amplify the release of a neurotransmitter and to accelerate the light-dark change in photosignals.

Adult↗
PubMed1999

Patch clamp recording of the responses to three bitter stimuli in mouse taste cells.

Although several pathways of bitter taste signal transduction have been proposed in taste cells, these mechanisms have not been elucidated in detail. To investigate the diversity of responses to bitter stimuli, we recorded the electrophysiological responses to quinine, denatonium and naringin using whole-cell patch clamp technique in isolated taste cells of C57BL/6J mice. Ten mM quinine induced depolarizing response under the current clamp mode, and inward current response under the voltage-clamp mode (holding potential -80 mV) using both K+ (with pseudo intracellular solution) and Cs+ (K+ was substituted by Cs+ in the pseudo intracellular solution) pipettes. However, when the K+ pipette was used, the membrane conductance was suppressed and activated in succession. On the other hand, the membrane conductance was only activated when the Cs+ pipette was used. Half to one mM denatonium induced depolarizing response under the current clamp mode, and outward current response under the voltage clamp mode with both pipettes. Using these pipettes, the membrane conductance was activated or suppressed in the individual case. Naringin-induced responses were not detected in these measurements. These electrophysiological recordings suggest that multiple transduction mechanisms are involved in bitter taste perception in mouse taste cells.

Animals↗
PubMed2006

Direct GABAergic and glycinergic inhibition of the substantia gelatinosa from the rostral ventromedial medulla revealed by in vivo patch-clamp analysis in rats.

Stimulation of the rostral ventromedial medulla (RVM) is believed to exert analgesic effects through the activation of the serotonergic system descending to the spinal dorsal horn; however, how nociceptive transmission is modulated by the descending system has not been fully clarified. To investigate the inhibitory mechanisms affected by the RVM, an in vivo patch-clamp technique was used to record IPSCs from the substantia gelatinosa (SG) of the spinal cord evoked by chemical (glutamate injection) and electrical stimulation (ES) of the RVM in adult rats. In the voltage-clamp mode, the RVM glutamate injection and RVM-ES produced an increase in both the frequency and amplitude of IPSCs in SG neurons that was not blocked by glutamate receptor antagonists. Serotonin receptor antagonists were unexpectedly without effect, but a GABAA receptor antagonist, bicuculline, or a glycine receptor antagonist, strychnine, completely suppressed the RVM stimulation-induced increase in IPSCs. The RVM-ES-evoked IPSCs showed fixed latency and no failure at 20 Hz stimuli with a conduction velocity of >3 m/s (3.1-20.7 m/s), suggesting descending monosynaptic GABAergic and/or glycinergic inputs from the RVM to the SG through myelinated fibers. In the current-clamp mode, action potentials elicited by noxious mechanical stimuli applied to the receptive field of the ipsilateral hindlimb were suppressed by the RVM-ES in more than half of the neurons tested (63%; 10 of 16). These findings suggest that the RVM-mediated antinociceptive effects on noxious inputs to the SG may be exerted preferentially by the direct GABAergic and glycinergic pathways to the SG.

Animals↗