Whole Cell Patch-Clamp Recordings of Rat Midbrain Dopaminergic Neurons Isolate a Sulphonylurea- and ATP-Sensitive Component of Potassium Currents Activated by Hypoxia

1998 ◽  
Vol 79 (3) ◽  
pp. 1239-1245 ◽  
Author(s):  
E. Guatteo ◽  
M. Federici ◽  
A. Siniscalchi ◽  
T. Knöpfel ◽  
N. B. Mercuri ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Dopaminergic Neurons ◽  
Outward Current ◽  
Potassium Currents ◽  
Whole Cell ◽  
Cell Patch Clamp ◽  
Whole Cell Patch Clamp ◽  
Midbrain Dopaminergic Neurons ◽  
Cytosolic Factors

Guatteo, E., M. Federici, A. Siniscalchi, T. Knöpfel, N. B. Mercuri, and G. Bernardi. Whole cell patch-clamp recordings of rat midbrain dopaminergic neurons isolate a sulphonylurea- and ATP-sensitive component of potassium currents activated by hypoxia. J. Neurophysiol. 79: 1239–1245, 1998. The effects of brief (2–4 min) hypoxia on presumed dopaminergic “principal” neurons of the rat ventral mesencephalon were investigated by using either intracellular or whole cell patch-clamp recordings in in vitro conditions. Under single-electrode voltage clamp, with sharp microelectrode ( V h −60 mV), a brief hypoxia caused an outward current (hypoOUT) of 110.2 ± 15.2 (SE) pA ( n = 18), which was followed by a posthypoxic outward current (posthypoOUT) of 149.6 ± 10.6 pA ( n = 18). Although the hypoOUT reversed at −83.7 ± 3.8 mV ( n = 18), the posthypoOUT did not reverse. The K+ ATP-blocking sulphonylureas tolbutamide (100 μM) and glibenclamide (30 μM), significantly reduced the peak of the hypoOUT by 47.6 ± 7.7% ( n = 16) and 54.18 ± 7.5% ( n = 3), respectively. In contrast, they did not affect the posthypoOUT. Extracellular barium (300 μM to 1 mM) almost abolished the hypoOUT, leaving the posthypoOUT unchanged. The large K+ channel blocker charybdotoxin (10–50 nM), depressed the hypoOUT after tolbutamide treatment. To investigate whether or not cytosolic factors might control the development of the hypoOUT, we dialyzed the principal neurons by patch-clamp recordings ( V h −60 mV). Under whole cell recordings hypoxia evoked an hypoOUT of 70.2 ± 14.5 pA that reversed polarity at −87.9 ± 5.1 mV ( n = 8). A small posthypoxic response was detected upon reoxygenation in a few neurons (4 out of 14). Three different sulphonylureas, tolbutamide (100 μM), glibenclamide (10–30 μM), and glipizide (100 nM) completely blocked the hypoOUT in patch-clamped neurons. The hypoOUT was also abolished by extracellular BaCl2 (300 μM). When the content of ATP in the dialyzate was raised from 2 to 10 mM no outward current/hyperpolarization was evoked by hypoxia. These data suggest that the hypoOUT, in principal neurons, is a complex response sustained by at least two barium-sensitive components: 1) an ATP-dependent, sulphonylurea-sensitive K+ conductance which could be isolated by the patch-clamp techniques and 2) a K+ conductance remaining after tolbutamide in intracellularly recorded neurons, which is sensitive to charybdotoxin and dependent on dialyzable cytosolic factors.

Whole Cell Recordings From Visualized Neurons in the Inner Laminae of the Functionally Intact Spinal Cord

2009 ◽  
Vol 102 (1) ◽  
pp. 590-597 ◽  
Author(s):  
Jason Dyck ◽  
Simon Gosgnach
Keyword(s):  
Neural Network ◽  
Spinal Cord ◽  
Locomotor Activity ◽  
Patch Clamp ◽  
Lumbar Spinal Cord ◽  
Whole Cell ◽  
Neuronal Populations ◽  
Cell Patch Clamp ◽  
Whole Cell Patch Clamp

The in vitro whole spinal cord preparation has been an invaluable tool for the study of the neural network that underlies walking because it provides a means of recording fictive locomotor activity following surgical and/or pharmacological manipulation. The recent use of molecular genetic techniques to identify discrete neuronal populations in the spinal cord and subsequent studies showing some of these populations to be involved in locomotor activity have been exciting developments that may lead to a better understanding of the structure and mechanism of function of this neural network. It would be of great benefit if the in vitro whole spinal cord preparation could be updated to allow for the direct targeting of genetically defined neuronal populations, allowing each to be characterized physiologically and anatomically. This report describes a new technique that enables the visualization of, and targeted whole cell patch-clamp recordings from, genetically defined populations of neurons while leaving connectivity largely intact. The key feature of this technique is a small notch cut in the lumbar spinal cord that reveals cells located in the intermediate laminae while leaving the ventral portion of the spinal cord—the region containing the locomotor neural network—untouched. Whole cell patch-clamp recordings demonstrate that these neurons are healthy and display large rhythmic depolarizations that are related to electroneurogram bursts recorded from ventral roots during fictive locomotion. Intracellular labeling demonstrates that this technique can also be used to map axonal projection patterns of neurons. We expect that this procedure will greatly facilitate electrophysiological and anatomical study of important neuronal populations that constitute neural networks throughout the CNS.

scholarly journals Die Konsequenzen der zeitspezifischen Deletion von Neuroligin 2 für die synaptische Übertragung, Plastizität und neuronale Exzitabilität im Gyrus Dentatus von adulten Mäusen

2021 ◽  
Author(s):  
◽  
Franziska Frank
Keyword(s):  
Patch Clamp ◽  
Western Blotting ◽  
Whole Cell ◽  
Klinische Relevanz ◽  
Cell Patch Clamp ◽  
Whole Cell Patch Clamp ◽  
Gyrus Dentatus

Eines der übergeordneten Ziele neurowissenschaftlicher Grundlagenforschung ist es, die Pathomechanismen neuropsychiatrischer Erkrankungsbilder besser zu verstehen. Als Erklärungsmodell für einige dieser Erkrankungen dient unter anderem ein gestörtes Verhältnis zwischen Exzitation und Inhibition im Gehirn. Synaptische Strukturproteine sind wichtige Modulatoren dieses Verhältnisses. Für eine unbeeinträchtigte inhibitorische synaptische Transmission spielt das postsynaptische Zelladhäsionsprotein Neuroligin 2 eine maßgebliche Rolle, um das Gleichgewicht zwischen Exzitation und Inhibition aufrechtzuerhalten. Neuroligin 2 ist an der inhibitorischen Synapse lokalisiert und beeinflusst die Entwicklung, Reifung und Funktion dieser Synapse. Die klinische Relevanz von Neuroligin 2 wurde bereits bei zahlreichen Erkrankungsbildern wie Schizophrenie, Depression oder Epilepsie im Rahmen von Studien nachgewiesen. Um das Verhältnis zwischen Exzitation und Inhibition in vivo sowie Mechanismen der synaptischen Übertragung und Plastizität zu untersuchen, hat sich die Ableitung von Feldpotentialen im Gyrus Dentatus des Hippocampus etabliert. Im Neuroligin 2 Knockout Mausmodell konnte bereits gezeigt werden, dass eine pränatale Deletion dieses Proteins eine stark erhöhte Erregbarkeit der Körnerzellen und eine verminderte GABAerge Netzwerkinhibition im Gyrus Dentatus in vivo zur Folge hat. Unklar blieb bisher, ob diese durch den konventionellen Neuroligin 2 Knockout (pränatal) hervorgerufenen Netzwerkveränderungen alleine auf das Fehlen dieses Proteins zurückzuführen sind oder durch eine zusätzliche Beeinträchtigung der Hirnentwicklung hervorgerufen werden. Ziel dieser Dissertation ist es deshalb, die Rolle von Neuroligin 2 im Gyrus Dentatus durch einen induzierten Knockout in adulten Mäusen (postnatal) unabhängig von einem möglichen Entwicklungseffekt zu klären. Dazu wurde im ersten methodischen Schritt dieser Dissertation durch orale Tamoxifen-Gabe eine zeitspezifische konditionale Eliminierung von Neuroligin 2 in genetisch modifizierten, adulten Mäusen erzielt. Im Anschluss an diese konditionale Eliminierung wurde die synaptische Transmission, Plastizität sowie neuronale Erregbarkeit von Körnerzellen im Gyrus Dentatus mittels elektrophysiologischer Experimente untersucht. Hierzu wurde zunächst der Tractus Perforans und die Körnerzellschicht durch stereotaktische Chirurgie in anästhesierten Mäusen lokalisiert. Anschließend wurde eine Stimulation des Tractus Perforans sowie eine Ableitung von Feldpotentialen im Gyrus Dentatus durchgeführt. Um die Erregbarkeit der Körnerzellen, die synaptische Transmission, Kurz- und Langzeitplastizität sowie Netzwerkinhibition im Gyrus Dentatus zu analysieren, wurden unterschiedliche Stimulationsprotokolle verwendet. Im Anschluss an die elektrophysiologischen Experimente wurden die Hippocampi beidseitig entnommen, konserviert und später einer Proteinquantifizierung von Neuroligin 2 mittels Western-Blotting unterzogen. Die Ergebnisse zeigten ein signifikant verringertes Proteinlevel von Neuroligin 2 auf 41,07% im Hippocampus von konditionalen Neuroligin 2 Knockout Mäusen. Unter dieser Reduktion von Neuroligin 2 in adulten Mäusen war die in vivo Erregbarkeit der Körnerzellen des Gyrus Dentatus sowie GABAerge Netzwerkinhibition weitgehend unbeeinträchtigt und die signifikanten Beobachtungen des konventionellen Knockout Modells ließen sich nicht reproduzieren. Aufgrund der unvollständigen Proteinreduktion lässt sich jedoch nicht abschließend beurteilen, ob die Restmenge den elektrophysiologischen Effekt kompensiert oder ob die im konventionellen Neuroligin 2 Knockout Modell beobachteten Effekte auf eine ausschließliche Rolle von Neuroligin 2 in der Hirnentwicklungsperiode zurückzuführen sind. Kürzlich veröffentlichte Daten zeigten allerdings, dass die postnatale Deletion von Neuroligin 2 in anderen Hirnregionen zu einer verminderten Netzwerkinhibition führt. Neben der hier verwendeten in vivo Methodik ist eine Ergänzung von Untersuchungen in nicht-anästhesierten Tieren sowie Messungen einzelner Zellen durch whole-cell patch-clamp Untersuchungen in vitro oder in vivo zu erwägen. Es sollte dabei auf eine konditionale Proteineliminierung geachtet werden, damit mögliche Kompensationsmechanismen weitgehend ausgeschlossen werden können. Eine weiterführende immunhistochemische Bildgebung der Hippocampuspräparate, wie sie im konventionellen Knockout durchgeführt wurde, könnte sich hierbei ebenso als aufschlussreich für die Funktion von Neuroligin 2 im Hippocampus des adulten Tieres erweisen.

Effects of Methylphenidate on the Membrane Potential and Current in Neurons of the Rat Locus Coeruleus

2002 ◽  
Vol 87 (3) ◽  
pp. 1206-1212 ◽  
Author(s):  
Masaru Ishimatsu ◽  
Yuri Kidani ◽  
Akira Tsuda ◽  
Takashi Akasu
Keyword(s):  
Membrane Potential ◽  
Patch Clamp ◽  
Locus Coeruleus ◽  
Outward Current ◽  
Inward Rectification ◽  
Spontaneous Firing ◽  
Patch Clamp Recording ◽  
Whole Cell ◽  
Cell Patch Clamp ◽  
Whole Cell Patch Clamp

Effects of methylphenidate (MPH), a therapeutic agent used in children presenting the attention deficit hyperactivity disorder (ADHD), on the membrane potential and current in neurons of the rat locus coeruleus (LC) were examined using intracellular and whole cell patch-clamp recording techniques. Application of MPH (30 μM) to artificial cerebrospinal fluid (ACSF) produced a hyperpolarizing response with amplitude of 12 ± 1 mV ( n = 29). Spontaneous firing of LC neurons was blocked during the MPH-induced hyperpolarization. Superfusion of LC neurons with ACSF containing 0 mM Ca2+ and 11 mM Mg2+ (Ca2+-free ACSF) produced a depolarizing response associated with an increase in spontaneous firing of the action potential. The MPH-induced hyperpolarization was blocked in Ca2+-free ACSF. Yohimbine (1 μM) and prazosin (10 μM), antagonists for α2 and α2B/2Creceptors, respectively, blocked the MPH-induced hyperpolarization in LC neurons. Tetrodotoxin (TTX, 1 μM) produced a partial depression of the MPH-induced hyperpolarization in LC neurons. Under the whole cell patch-clamp condition, MPH (30–300 μM) produced an outward current ( I MPH) with amplitude of 110 ± 6 pA ( n = 17) in LC neurons. The I MPH was blocked by Co2+ (1 mM). During prolonged application of MPH (300 μM for 45 min), the hyperpolarization gradually decreased in the amplitude and eventually disappeared, possibly because of depression of norepinephrine (NE) release from noradrenergic nerve terminals. At a low concentration (1 μM), MPH produced no outward current but consistently enhanced the outward current induced by NE. These results suggest that the MPH-induced response is mediated by NE via α2B/2C-adrenoceptors in LC neurons. I MPH was associated with an increase in the membrane conductance of LC neurons. The I MPH reversed its polarity at −102 ± 6 mV ( n = 8) in the ACSF. The reversal potential of I MPH was changed by 54 mV per decade change in the external K+ concentration. Current-voltage relationship showed that the I MPH exhibited inward rectification. Ba2+ (100 μM) suppressed the amplitude and the inward rectification of the I MPH.These results suggest that the I MPH is produced by activation of inward rectifier K+channels in LC neurons.

scholarly journals Multiple two-photon targeted whole-cell patch-clamp recordings from monosynaptically connected neurons in vivo

2019 ◽  
Author(s):  
Jean-Sébastien Jouhanneau ◽  
James F.A. Poulet
Keyword(s):  
Patch Clamp ◽  
Whole Cell ◽  
Two Photon ◽  
Cell Patch Clamp ◽  
Whole Cell Patch Clamp ◽  
Brain States ◽  
Post Hoc ◽  
Whole Cell Recordings

AbstractAlthough we know a great deal about monosynaptic connectivity, transmission and integration in the mammalian nervous system from in vitro studies, very little is known in vivo. This is partly because it is technically difficult to evoke action potentials and simultaneously record small amplitude subthreshold responses in closely (< 150 µm) located pairs of neurons. To address this, we have developed in vivo two-photon targeted multiple (2 – 4) whole-cell patch clamp recordings of nearby neurons in superficial cortical layers 1 to 3. Here we describe a step-by-step guide to this approach in the anesthetised mouse primary somatosensory cortex, including: the design of the setup, surgery, preparation of pipettes, targeting and acquisition of multiple whole-cell recordings, as well as in vivo and post-hoc histology. The procedure takes ∼ 4 hours from start of surgery to end of recording and allows examinations both into the electrophysiological features of unitary excitatory and inhibitory monosynaptic inputs during different brain states as well as the synaptic mechanisms of correlated neuronal activity.

Whole-cell Cl− currents in a human peripheral airway epithelial cell line

1993 ◽  
Vol 71 (9) ◽  
pp. 662-670 ◽  
Author(s):  
Xiaodong Wang ◽  
Ludwik Fedorko ◽  
Yoshinori Marunaka ◽  
Hugh O'Brodovich
Keyword(s):  
Cyclic Amp ◽  
Patch Clamp ◽  
Cell Line ◽  
Pertussis Toxin ◽  
Outward Current ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Cell Patch Clamp ◽  
Whole Cell Patch Clamp ◽  
Peripheral Airway

We have used the whole-cell patch-clamp technique to identify and characterize Cl− currents in a cell line derived from human peripheral airway epithelium (NCI-H-441-4). The permeability sequence and relative selectivity for different anions was Br− (1.4) ~ I− (1.3) > Cl− (1.0) > F− (0.6) > gluconate (0.4) > glutamate (0.2). The current–voltage relationship displayed rectification in the outward direction. Diphenylamine-2-carboxylate (10−4 M) applied intracellularly blocked the outward-rectified current, while extracellularly applied diphenylamine-2-carboxylate had no effect on Cl− current. This current was also blocked by extracellularly applied 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB), with an estimated IC50 of 15.2 μM. Dibutyryl-cyclic AMP (10−4 M) increased outward current, whereas pretreatment with 100 ng/mL pertussis toxin almost completely abolished the Cl− current. Pertussis toxin inhibition of this current could be partially reversed by dialysis of the cell interior with the activated αi–2 subunit of Gi protein. This cell line provides an opportunity to study directly the regulation of Cl− channels in cells derived from the peripheral human lung airways.Key words: chloride secretion, whole-cell patch clamp, GTP binding protein, cyclic AMP, pertussis toxins, 5-nitro-2-(3-phenylpropylamino)benzoate, diphenylamine-2-carboxylate, cell line H441.

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