scholarly journals Muscarinic activation of ionic currents measured by a new whole-cell recording method.

1988 ◽  
Vol 92 (2) ◽  
pp. 145-159 ◽  
Author(s):  
R Horn ◽  
A Marty
Keyword(s):  
Membrane Conductance ◽  
Ionic Currents ◽  
Response Curves ◽  
Whole Cell ◽  
Whole Cell Recording ◽  
Muscarinic Response ◽  
Cell Recording ◽  
K Current ◽  
A Cell ◽  
K Currents

A new method is described as an alternative to whole-cell recording in order to prevent "wash-out" of the muscarinic response to acetylcholine (ACh) in rat lacrimal gland cells. The membrane of a cell-attached patch is permeabilized by nystatin in the patch pipette, thus providing electrical continuity between the pipette and the cytoplasm of the cell without the loss or alteration of cytoplasmic compounds necessary for the maintenance of the response to ACh. With normal whole-cell recording in these cells, the response to ACh, seen as the activation of Ca-activated K and Cl currents, lasts for approximately 5 min. With the nystatin method, the response is not diminished after 1 h. Nystatin, applied extracellularly, is shown to cause a rapid and reversible increase of membrane conductance to cations. In the absence of wash-out, we were able to obtain dose-response curves for the effect of ACh on Ca-activated K currents. An increase of [ACh] caused an increase in the K current, with apparent saturation at concentrations above approximately 1 microM ACh. The delay between ACh application and the activation of K current was inversely related to [ACh] and reached a minimum value of 0.7-1.0 s at high [ACh].

scholarly journals Chemosensory responses in isolated olfactory receptor neurons from Necturus maculosus.

1992 ◽  
Vol 99 (3) ◽  
pp. 415-433 ◽  
Author(s):  
V E Dionne
Keyword(s):  
Olfactory Receptor ◽  
Membrane Conductance ◽  
Input Resistance ◽  
Primary Response ◽  
Olfactory Receptor Neurons ◽  
Whole Cell ◽  
Whole Cell Recording ◽  
Necturus Maculosus ◽  
Cell Recording ◽  
Receptor Neurons

Olfactory receptor neurons were isolated without enzymes from the mudpuppy, Necturus maculosus, and tested for chemosensitivity. The cells responded to odorants with changes in firing frequency and alterations in excitability that were detected with tight-seal patch electrodes using on-cell and whole-cell recording conditions. Chemosensitive cells exhibited two primary response characteristics: excitation and inhibition. Both types of primary response were observed in different cells stimulated by mixtures of amino acids as well as by the single compound L-alanine, suggesting that there may be more than one transduction pathway for some odorants. Using the normal whole-cell recording method, the chemosensitivity of competent cells washed out rapidly; a resistive whole-cell method was used to record odorant responses under current-clamp conditions. In response to chemical stimulation, excitability appeared to be modulated in several different ways in different cells: odorants induced hyperpolarizing or depolarizing receptor potentials, elicited or inhibited transient, rhythmic generator potentials, and altered excitability without changing the membrane potential or input resistance. These effects suggest that olfactory transduction is mediated through at least three different pathways with effects on four or more components of the membrane conductance. Polychotomous pathways such as these may be important for odor discrimination and for sharpening the "odor image" generated in the olfactory epithelium.

Acetylcholine potentiates acetylcholine-induced increases in K+ current in cat atrial myocytes

1995 ◽  
Vol 268 (3) ◽  
pp. H1313-H1321 ◽  
Author(s):  
Y. G. Wang ◽  
S. L. Lipsius
Keyword(s):  
Reversal Potential ◽  
Membrane Conductance ◽  
K Channel ◽  
Channel Blockers ◽  
Atrial Myocytes ◽  
Initial Exposure ◽  
Recovery Interval ◽  
Cell Recording ◽  
K Current ◽  
K Currents

A nystatin-perforated patch whole cell recording method was used to study the effects of acetylcholine (ACh) on ACh-induced K+ currents in atrial myocytes isolated from cat hearts. The general protocol involved an initial 4-min exposure to ACh (ACh1), followed by a 4-min washout in ACh-free Tyrode solution and then a second 4-min ACh exposure (ACh2). Voltage ramps (40 mV/s) between -130 and +30 mV were used to assess changes in total membrane conductance. ACh2 (10 microM) induced an increase in K+ conductance that was significantly larger than that induced by ACh1 (10 microM) at voltages both negative and positive to the reversal potential. The potentiated current induced by ACh2 reversed at about -80 mV and inwardly rectified at voltages positive to the reversal potential. External Ba2+ (5 mM) or tetraethylammonium (10 mM) abolished all ACh2-induced increases in membrane conductance. The sensitivity to K+ channel blockers, reversal potential, and the rectifying properties indicate that the current potentiated by ACh2 is a K+ current. Atropine (1 microM) blocked all effects of ACh on K+ currents. Potentiation of K+ current by ACh2 required 1) ACh1 concentrations > or = 1 microM, 2) ACh1 duration > or = 2 min, and 3) recovery interval > or = 2 min. We conclude that an initial exposure to ACh potentiates subsequent ACh-induced increases in K+ current. ACh-induced potentiation depends on the concentration and duration of the initial ACh exposure and the recovery interval between consecutive ACh exposures.(ABSTRACT TRUNCATED AT 250 WORDS)

Membrane currents and cholinergic regulation of K+ current in esophageal smooth muscle cells

1990 ◽  
Vol 258 (5) ◽  
pp. G794-G802 ◽  
Author(s):  
S. M. Sims ◽  
M. B. Vivaudou ◽  
C. Hillemeier ◽  
P. Biancani ◽  
J. V. Walsh ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Membrane Currents ◽  
Equilibrium Potential ◽  
Whole Cell ◽  
Inward Current ◽  
Whole Cell Recording ◽  
Cell Recording ◽  
K Current

The tight-seal whole cell recording technique with patch pipettes was used to study membrane currents of smooth muscle cells freshly dissociated from the esophagus of cats. Under voltage clamp with K+ in the pipette, depolarizing commands elicited an initial inward current followed by a transient outward current that peaked and then declined to reveal spontaneous outward currents (SOCs). SOCs were evident at -60 mV and more positive potentials. The reversal of SOCs at the K+ equilibrium potential and their suppression by tetraethylammonium chloride lead to the conclusion that they represent the activity of K+ channels. Acetylcholine (ACh) caused reversible contraction of these cells and had two successive effects on membrane currents, causing transient activation of K+ current followed by suppression of SOCs. Both of these effects were blocked by atropine. Consistent with these observations, in current clamp, ACh caused a transient hyperpolarization followed by depolarization. The inward current activated by depolarization was blocked by external Cd2+, consistent with the inward current being a voltage-activated calcium current. Two types of Ca2+ current could be distinguished on the basis of voltage-activation range, time course of inactivation and "run-down" during whole cell recording.

Perforated-patch recording does not enhance effect of 3-isobutyl-1-methylxanthine on cardiac calcium current

1994 ◽  
Vol 266 (6) ◽  
pp. C1619-C1627 ◽  
Author(s):  
A. Kawamura ◽  
G. M. Wahler
Keyword(s):  
Ventricular Myocytes ◽  
Cell Voltage ◽  
Rapid Decline ◽  
Response Curves ◽  
Whole Cell ◽  
Perforated Patch ◽  
Cell Responses ◽  
Whole Cell Recording ◽  
Cell Recording ◽  
Beta Adrenergic Agonist

Conventional whole cell voltage-clamp recording results in washout of the cardiac Ca2+ current (ICa) response to the beta-adrenergic agonist isoproterenol (Iso), for reasons which are not clear. When dose-response curves for the phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (IBMX) were compared using perforated-patch vs. conventional whole cell recording in guinea pig ventricular myocytes, the conventional whole cell IBMX responses were unexpectedly larger than the perforated-patch responses. Furthermore, during conventional whole cell recording the response to repeated application of Iso declined rapidly, whereas the IBMX response initially increased and then declined. When pipette [Ca2+] was increased to 10(-7) M, conventional whole cell responses to 300 microM IBMX and 10(-9) M Iso were identical to perforated-patch responses. Thus loss of the Iso response during conventional whole cell recording seems to not be solely due to a washout of some constituent of the adenosine 3',5'-cyclic monophosphate pathway. We suggest that unphysiological intracellular [Ca2+] enhances the relative PDE activity and that this contributes to the rapid decline of the Iso response and the initial enhancement of the IBMX response.

Phenylephrine suppresses outward K+ currents in rat atrial myocytes

1996 ◽  
Vol 271 (3) ◽  
pp. H937-H946 ◽  
Author(s):  
D. R. Van Wagoner ◽  
M. Kirian ◽  
M. Lamorgese
Keyword(s):  
Current Component ◽  
Atrial Myocytes ◽  
Adrenergic Agonist ◽  
Whole Cell ◽  
K Channels ◽  
Recovery From Inactivation ◽  
Cell Recording ◽  
K Current ◽  
Kinetics Of ◽  
K Currents

The modulation of whole cell K+ currents by the alpha 1-adrenergic agonist, phenylephrine, was studied in isolated rat atrial myocytes by use of perforated-patch whole cell recording techniques. The out ward K+ current in these myocytes consists of two inactivating components (iK,f and iK,s), which differ in the kinetics of inactivation and recovery from inactivation, and a noninactivating component, (iK,ss). Superfusion of these myocytes with 10 microM phenylephrine caused a rapid suppression of iK,ss, with little effect on the other current components. This effect of phenylephrine could be mimicked by exogenous application of 1,2-dioctanoyl-sn-glycerol (20 microM), a membrane-permeant diacylglycerol analogue; however, it was clearly distinct from the effect of 5 nM alpha-dendrotoxin, which selectively suppressed the slowly inactivating current component, iK,s, while having no effect on iK,ss. At a dose of 50 microM, phenylephrine also suppressed iK,s. There was no significant effect of phenylephrine (10 or 50 microM) or alpha-dendrotoxin (5 nM) on the rapidly inactivating current component, iK,f. The kinetic and pharmacological differences between these current components suggest that they represent the activity of distinct K+ channels.

Whole cell recording from neurons in slices of reptilian and mammalian cerebral cortex

1989 ◽  
Vol 30 (3) ◽  
pp. 203-210 ◽  
Author(s):  
Mark G. Blanton ◽  
Joseph J. Lo Turco ◽  
Arnold R. Kriegstein
Keyword(s):  
Cerebral Cortex ◽  
Whole Cell ◽  
Whole Cell Recording ◽  
Cell Recording

scholarly journals Knockouts reveal overlapping functions of M2 and M4 muscarinic receptors and evidence for a local glutamatergic circuit within the laterodorsal tegmental nucleus

2012 ◽  
Vol 108 (10) ◽  
pp. 2751-2766 ◽  
Author(s):  
Kristi A. Kohlmeier ◽  
Masaru Ishibashi ◽  
Jürgen Wess ◽  
Martha E. Bickford ◽  
Christopher S. Leonard
Keyword(s):  
Acetylcholine Receptors ◽  
Brain Slices ◽  
Cholinergic Neurons ◽  
Muscarinic Acetylcholine Receptors ◽  
Feedback Signal ◽  
Laterodorsal Tegmental Nucleus ◽  
Membrane Hyperpolarization ◽  
Whole Cell ◽  
Whole Cell Recording ◽  
Cell Recording

Cholinergic neurons in the laterodorsal tegmental (LDT) and peduncolopontine tegmental (PPT) nuclei regulate reward, arousal, and sensory gating via major projections to midbrain dopamine regions, the thalamus, and pontine targets. Muscarinic acetylcholine receptors (mAChRs) on LDT neurons produce a membrane hyperpolarization and inhibit spike-evoked Ca2+ transients. Pharmacological studies suggest M2 mAChRs are involved, but the role of these and other localized mAChRs (M1--M4) has not been definitively tested. To identify the underlying receptors and to circumvent the limited receptor selectivity of available mAChR ligands, we used light- and electron-immunomicroscopy and whole cell recording with Ca2+ imaging in brain slices from knockout mice constitutively lacking either M2, M4, or both mAChRs. Immunomicroscopy findings support a role for M2 mAChRs, since cholinergic and noncholinergic LDT and pedunculopontine tegmental neurons contain M2-specific immunoreactivity. However, whole cell recording revealed that the presence of either M2 or M4 mAChRs was sufficient, and that the presence of at least one of these receptors was required for these carbachol actions. Moreover, in the absence of M2 and M4 mAChRs, carbachol elicited both direct excitation and barrages of spontaneous excitatory postsynaptic potentials (sEPSPs) in cholinergic LDT neurons mediated by M1 and/or M3 mAChRs. Focal carbachol application to surgically reduced slices suggest that local glutamatergic neurons are a source of these sEPSPs. Finally, neither direct nor indirect excitation were knockout artifacts, since each was detected in wild-type slices, although sEPSP barrages were delayed, suggesting M2 and M4 receptors normally delay excitation of glutamatergic inputs. Collectively, our findings indicate that multiple mAChRs coordinate cholinergic outflow from the LDT in an unexpectedly complex manner. An intriguing possibility is that a local circuit transforms LDT muscarinic inputs from a negative feedback signal for transient inputs into positive feedback for persistent inputs to facilitate different firing patterns across behavioral states.

scholarly journals Compensation of physiological motion enables high-yield whole-cell recording in vivo

2020 ◽  
Author(s):  
William M. Stoy ◽  
Bo Yang ◽  
Ali Kight ◽  
Nathaniel C. Wright ◽  
Peter Y. Borden ◽  
...  
Keyword(s):  
Single Cells ◽  
Strong Dependence ◽  
High Yield ◽  
Patch Clamp Recording ◽  
Gold Standard Method ◽  
Whole Cell ◽  
Whole Cell Recording ◽  
Cell Recording ◽  
Living Mouse

1.1.1AbstractWhole-cell patch-clamp recording in vivo is the gold-standard method for measuring subthreshold electrophysiology from single cells during behavioural tasks, sensory stimulations, and optogenetic manipulation. However, these recordings require a tight, gigaohm resistance, seal between a glass pipette electrode’s aperture and a cell’s membrane. These seals are difficult to form, especially in vivo, in part because of a strong dependence on the distance between the pipette aperture and cell membrane. We elucidate and utilize this dependency to develop an autonomous method for placement and synchronization of pipette’s tip aperture to the membrane of a nearby, moving neuron, which enables high-yield seal formation and subsequent recordings in the deep in the brain of the living mouse, in the thalamus. This synchronization procedure nearly doubles the reported gigaseal yield in the thalamus (>3 mm below the pial surface) from 26% (n=17/64) to 48% (n=32/66). Whole-cell recording yield improved from 10% (n = 9/88) to 24% (n=18/76) when motion compensation was used during the gigaseal formation. As an example of its application, we utilized this system to investigate the role of the sensory environment and ventral posterior medial region (VPM) projection synchrony on intracellular dynamics in the barrel cortex. This method results in substantially greater subcortical whole-cell recording yield than previously reported and thus makes pan-brain whole-cell electrophysiology practical in the living mouse brain.

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