وقد تم تمويل هذا العمل من قبل مجلس البحوث السويدية ، وجامعة أوبسالا فريدريك وانغريد مؤسسة Thurings. عقدت ZJ زمالة ما بعد الدكتوراه من EXODIAB وSvenska Sällskapet لForskning Medicinsk. نشكر فرانك لي للحصول على المساعدة التقنية.

<ol>
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الإعلان عن أي تضارب في المصالح.

التيار خلية كاملة تمثل مجموعة متكاملة من التيارات التي تم إنشاؤها بواسطة القنوات النشطة في الخلية. وعلم الصيدلة والنفاذية كلوريد محددة من القنوات GABAA يجعل من الممكن تحديد التيارات المرتبطة بهذه القنوات. طبيعة عابرة لتفعيل قنوات متشابك وتنشيط منشط للقنوات extrasynaptic يسمح السهل التفريق بين هؤلاء السكان اثنين من قنوات GABAA أعرب في الخلايا العصبية. ومع ذلك ، لا يمكن إلا على قناة واحدة مع تسجيلات دراسة واحدة من الخصائص الحركية للجزيئات القناة التي تولد تيارات منشط. على عكس القنوات التي تنشط داخل متشابك 100 ميكرو ثانية ، يتم تنشيط قنوات GABAA منشط مع الكمون في نطاق دقائق من الوقت يتم تطبيق 4،7،15،16،17،18 GABA. لا يمكن تأجيل تفعيل هذه النتائج في تلك القنوات قناة خصائص كثيرة يمكن دراستها باستخدام خلية كاملة التسجيلات. ويمكن في مستوى واحد قناة يتم التعرف على مختلف المجموعات الفرعية من السكان من القنوات GABAA على أساس اختلاف الخصائص الفنية والدوائية. وهكذا ، عن طريق اتخاذ ما ميزة التصحيح المختلفة ، المشبك تكوينات لهذا العرض ، فمن الممكن للدراسة في التفاصيل الدالة الجزيئية وعلم الصيدلة من القنوات التي تنشط GABA التي تولد تيارات منشط ومتشابك في الخلايا العصبية.

العديد من التصحيح ، المشبك الأجهزة والبرمجيات ونظم متوفرة وتعمل به جميع المبادئ الأساسية نفسها. في مختبرنا نستخدم مكبرات الصوت Axoclamp والبرامج PClamp (الأجهزة الجزيئية ، الولايات المتحدة) لتسجيل وتحليل تجاربنا. ماركة أخرى شعبية HEKA مكبرات الصوت والبرمجيات (HEKA الكترونيك ، ألمانيا) Microforge : Narishige MF - 830 (اليابان) ؛ Vibrotome : على سبيل المثال لايكا VT1200S (ألمانيا) أو آلات Campden vibrotome (المملكة المتحدة) ؛ مجهر : نيكون الكسوف E600FN ؛ حمام المياه ؛ Micromanipulators : العرف وmicromanipulators Narishige (اليابان) ؛ بولير : DMZ - العالمي (ألمانيا) أو Narishige PC - 10 (اليابان). الزجاج الكهربائي : GC150F - 15 (جهاز هارفارد ، المملكة المتحدة).

gaba-activated single-channel and tonic currents in rat brain slices

وGABA A قنوات موجودة في جميع الخلايا العصبية وتوجد في كل من نقاط الاشتباك العصبي وخارج نقاط الاشتباك العصبي ، حيث أنها تولد تيارات طوري ومنشط ، على التوالي 4،5،6،7 A GABA والقناة هي القناة pentameric كلوريد GABA بوابات. يتم تجميع مفارز قناة إلى 8 أسرة (α1 - 6 ، β1 - 3 ، γ1 - 3 ، δ ، ε ، θ ، وπ ρ). alphas اثنين ، الجيدات مرتين والثالثة 3 الوحيدات شكل القناة وظيفية 8. من خلال الجمع بين الدراسات الفرعية نوع محدد الجزيئات على قناة واحدة مع GABA تنشيط الدراسات بما في ذلك جميع سكان GABA A قنوات في الخلايا العصبية يصبح من الممكن فهم الآلية الأساسية لتثبيط الخلايا العصبية وكيف هو منظم من قبل وكلاء الدوائية. نستخدم تقنية التصحيح ، المشبك 9،10 لدراسة الخصائص الفنية للقنوات وغابا في الخلايا العصبية على قيد الحياة في شرائح الدماغ الحصين وتسجيل قناة واحدة والتيارات خلية كاملة. علينا مواصلة دراسة كيفية تأثر قنوات مختلفة من تركيزات GABA ، والمخدرات ، وعوامل أخرى داخل وخارج الخلية. وصف تفصيلي للالنظري والعملي للطريقة التصحيح ، يرجى الاطلاع على المشبك واحدة القناة تسجيلات تاليف Sakman B و E Neher 10.

Watch this Scientific Journal Video about GABA-activated Single-channel and Tonic Currents in Rat Brain Slices at JoVE.com

GABA-activated Single-channel and Tonic Currents in Rat Brain Slices

نستخدم تقنية التصحيح ، المشبك لقياس GABA تنشيط المفرد قناة التيارات (GABAA القنوات ، المستقبلات GABAA) والتيارات متشابك ومنشط التي تولدها في الخلايا العصبية. تفعيل قنوات إنقاص استثارة الخلايا العصبية في الصحة والمرض<sup&gt; 1،2،3،4</sup&gt;.

GABA تنشيط التيارات قناة واحدة وتونيك في شرائح الدماغ الجرذ

The GABAA channels are present in all neurons and are located both at synapses and outside of synapses where they generate phasic and tonic currents, ...

gaba-activated-single-channel-and-tonic-currents-in-rat-brain-slices

Research

JoVE Journal

Neuroscience

25.1K Views.  Uppsala University, Sweden. نستخدم تقنية التصحيح ، المشبك لقياس GABA تنشيط المفرد قناة التيارات (GABAA القنوات ، المستقبلات GABAA) والتيارات متشابك ومنشط التي تولدها في الخلايا العصبية. تفعيل قنوات إنقاص استثارة الخلايا العصبية في الصحة والمرض 1،2،3،4.

Video: GABA-activated Single-channel and Tonic Currents in Rat Brain Slices

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

excitotoxic المواد تحفيز الدماغ باعتبارها Microslices<em&gt; في المختبر</em&gt; نموذج من السكتة الدماغية

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Medicine

Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal population (10-20%), but show a high level of physiological, morphological, and neurochemical heterogeneity, reflecting their diverse functions. Therefore, investigation of interneurons provides important insights into the organization principles and function of neuronal circuits. This, however, requires an integrated physiological and neuroanatomical approach for the selection and identification of individual interneuron types. Whole-cell patch-clamp recording from acute brain slices of transgenic animals, expressing fluorescent proteins under the promoters of interneuron-specific markers, provides an efficient method to target and electrophysiologically characterize intrinsic and synaptic properties of specific interneuron types. Combined with intracellular dye labeling, this approach can be extended with post-hoc morphological and immunocytochemical analysis, enabling systematic identification of recorded neurons. These methods can be tailored to suit a broad range of scientific questions regarding functional properties of diverse types of cortical neurons.

Cortical networks are controlled by a small, but diverse set of inhibitory interneurons. Functional investigation of interneurons therefore requires targeted recording and rigorous identification. Described here is a combined approach involving whole-cell recordings from single or synaptically-coupled pairs of neurons with intracellular labeling, post-hoc morphological and immunocytochemical analysis.

خلية كاملة تسجيلات التصحيح، المشبك من Morphologically- وحددت Neurochemically-الحصين Interneurons

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal ...

Generation of Local CA1 &#947; Oscillations by Tetanic Stimulation

Neuronal network oscillations are important features of brain activity in health and disease and can be modulated by a range of clinically used drugs. A protocol is provided to generate a model for studying CA1 &#947; oscillations (20 - 80 Hz). These &#947; oscillations are stable for at least 30 min&#160;and depend upon excitatory and inhibitory synaptic activity in addition to activation of pacemaker currents. Tetanically stimulated oscillations have a number of reproducible and easily quantifiable characteristics including spike count, oscillation duration, latency and frequency that report upon the network state. The advantages of the electrically stimulated oscillations include stability, reproducibility and episodic acquisition enabling robust characterization of network function. This model of CA1 &#947; oscillations can be used to study cellular mechanisms and to systematically investigate how neuronal network activity is altered in disease and by drugs. Disease state pharmacology can be readily incorporated by the use of brain slices from genetically modified or interventional animal models to enable selection of drugs that specifically target disease mechanisms.

Oscillations are fundamental network properties and are modulated by disease and drugs. Studying brain-slice oscillations allows characterization of isolated networks under controlled conditions. Protocols are provided for the preparation of acute brain slices for evoking CA1 &#947; oscillations.

جيل من التذبذبات CA1 γ المحلية من خلال تحفيز كزازي

Neuronal network oscillations are important features of brain activity in health and disease and can be modulated by a range of clinically used drugs. A ...

Whole-cell Currents Induced by Puff Application of GABA in Brain Slices

Pharmacological administration is commonly used when conducting whole-cell patch-clamp recording in brain slices. One of the best methods of drug application during electrophysiological recording is the puff technique, which can be used to study the effect of pharmacological reagents on neuronal activities in brain slices. The greatest advantage of puff application is that the drug concentration around the recording site increases rapidly, thus preventing desensitization of membrane receptors. Successful use of puff application involves careful attention to the following elements: the concentration of the drug, the parameters of the puff micropipette, the distance between the tip of a puff micropipette and the neuron recorded, and the duration and pressure driving the puff (pounds per square inch, psi). This article describes a step-by-step procedure for recording whole-cell currents induced by puffing gamma-aminobutyric acid (GABA) onto a neuron of a prefrontal cortical slice. Notably, the same procedure can be applied with minor modifications to other brain areas such as the hippocampus and the striatum, and to different preparations, such as cell cultures.

We describe the puff technique, by which pharmacological reagents can be administered during whole-cell patch-clamp recording, and highlight various aspects of the features that are crucial for its success.

كامل الخلية التيارات الناجمة عن تطبيق نفخة GABA في الدماغ شرائح

Pharmacological administration is commonly used when conducting whole-cell patch-clamp recording in brain slices. One of the best methods of drug ...

Recording and Modulation of Epileptiform Activity in Rodent Brain Slices Coupled to Microelectrode Arrays

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) is a promising approach when pharmacological treatment fails or neurosurgery is not recommended. Acute brain slices coupled to microelectrode arrays (MEAs) represent a valuable tool to study neuronal network interactions and their modulation by electrical stimulation. As compared to conventional extracellular recording techniques, they provide the added advantages of a greater number of observation points and a known inter-electrode distance, which allow studying the propagation path and speed of electrophysiological signals. However, tissue oxygenation may be greatly impaired during MEA recording, requiring a high perfusion rate, which comes at the cost of decreased signal-to-noise ratio and higher oscillations in the experimental temperature. Electrical stimulation further stresses the brain tissue, making it difficult to pursue prolonged recording/stimulation epochs. Moreover, electrical modulation of brain slice activity needs to target specific structures/pathways within the brain slice, requiring that electrode mapping be easily and quickly performed live during the experiment. Here, we illustrate how to perform the recording and electrical modulation of 4-aminopyridine (4AP)-induced epileptiform activity in rodent brain slices using planar MEAs. We show that the brain tissue obtained from mice outperforms rat brain tissue and is thus better suited for MEA experiments. This protocol guarantees the generation and maintenance of a stable epileptiform pattern that faithfully reproduces the electrophysiological features observed with conventional field potential recording, persists for several hours, and outlasts sustained electrical stimulation for prolonged epochs. Tissue viability throughout the experiment is achieved thanks to the use of a small-volume custom recording chamber allowing for laminar flow and quick solution exchange even at low (1 mL/min) perfusion rates. Quick MEA mapping for real-time monitoring and selection of stimulating electrodes is performed by a custom graphic user interface (GUI).

We illustrate how to perform recording and electrical modulation of 4-aminopyridine-induced epileptiform activity in rodent brain slices using microelectrode arrays. A custom recording chamber maintains tissue viability throughout prolonged experimental sessions. Live electrode mapping and selection of stimulating pairs are performed by a custom graphical user interface.

تسجيل وتعديل النشاط صرعية في الدماغ القوارض شرائح بالإضافة إلى صفائف ميكروليكترودي

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) ...

Methods for the Discovery of Novel Compounds Modulating a Gamma-Aminobutyric Acid Receptor Type A Neurotransmission

This manuscript presents a step-by-step protocol for screening compounds at gamma-aminobutyric acid type A (GABAA) receptors and its use towards the identification of novel molecules active in preclinical assays from an in vitro recombinant receptor to their pharmacological effects at native receptors in rodent brain slices. For compounds binding at the benzodiazepine site of the receptor, the first step is to set up a primary screen that consists of developing radioligand binding assays on cell membranes expressing the major GABAA subtypes. Then, taking advantage of the heterologous expression of rodent and human GABAA receptors in Xenopus oocytes or HEK 293 cells, it is possible to explore, in electrophysiological assays, the physiological properties of the different receptor subtypes and the pharmacological properties of the identified compounds. The Xenopus oocyte system will be presented here, starting with the isolation of the oocytes and their microinjection with different mRNAs, up to the pharmacological characterization using two-electrode voltage clamps. Finally, recordings conducted in rodent brain slices will be described that are used as a secondary physiological test to assess the activity of molecules at their native receptors in a well-defined neuronal circuit. Extracellular recordings using population responses of multiple neurons are demonstrated together with the drug application.

Here, we present protocols to discover compounds active at GABAA receptors, from the binding to the physiology and pharmacology.

اكتب أساليب لاكتشاف مركبات الرواية تحوير مستقبلات حمض غاما أمينوبوتيريك – كبيرة

This manuscript presents a step-by-step protocol for screening compounds at gamma-aminobutyric acid type A (GABAA) receptors and its use towards the ...

Generation and On-Demand Initiation of Acute Ictal Activity in Rodent and Human Tissue

Controlling seizures remains a challenging issue for the medical community. To make progress, researchers need a way to extensively study seizure dynamics and investigate its underlying mechanisms. Acute seizure models are convenient, offer the ability to perform electrophysiological recordings, and can generate a large volume of electrographic seizure-like (ictal) events. The promising findings from acute seizure models can then be advanced to chronic epilepsy models and clinical trials. Thus, studying seizures in acute models that faithfully replicate the electrographic and dynamical signatures of a clinical seizure will be essential for making clinically relevant findings. Studying ictal events in acute seizure models prepared from human tissue is also important for making findings that are clinically relevant. The key focus in this paper is on the cortical 4-AP model due to its versatility in generating ictal events in both in vivo and in vitro studies, as well as in both mouse and human tissue. The methods in this paper will also describe an alternative method of seizure induction using the Zero-Mg2+ model and provide a detailed overview of the advantages and limitations of the epileptiform-like activity generated in the different acute seizure models. Moreover, by taking advantage of commercially available optogenetic mouse strains, a brief (30 ms) light pulse can be used to trigger an ictal event identical to those occurring spontaneously. Similarly, 30 - 100 ms puffs of neurotransmitters (Gamma-Amino Butyric Acid or glutamate) can be applied to the human tissue to trigger ictal events that are identical to those occurring spontaneously. The ability to trigger ictal events on-demand in acute seizure models offers the newfound ability to observe the exact sequence of events that underlie seizure initiation dynamics and efficiently evaluate potential anti-seizure therapies.

Acute seizure models are important for studying the mechanisms underlying epileptiform events. Furthermore, the ability to generate epileptiform events on-demand provides a highly efficient method to study the exact sequence of events underlying their initiation. Here, we describe the acute 4-aminopyridine cortical seizure models established in mouse and human tissue.

بدء نشاط Ictal الحادة في القوارض والأنسجة البشرية الجيل وبناء على الطلب

Controlling seizures remains a challenging issue for the medical community. To make progress, researchers need a way to extensively study seizure dynamics ...

Horizontal Hippocampal Slices of the Mouse Brain

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as well as the manifestation of severe brain disorders, including Alzheimer&#8217;s disease and epilepsy. The hippocampus receives a high degree of intra- and inter-connectivity, securing a proper communication with internal and external brain structures. This connectivity is accomplished via different informational flows in the form of fiber pathways. Brain slices are a frequently used methodology when exploring neurophysiological functions of the hippocampus. Hippocampal brain slices can be used for several different applications, including electrophysiological recordings, light microscopic measurements as well as several molecular biological and histochemical techniques. Therefore, brain slices represent an ideal model system to assess protein functions, to investigate pathophysiological processes involved in neurological disorders as well as for drug discovery purposes.
There exist several different ways of slice preparations. Brain slice preparations with a vibratome allow a better preservation of the tissue structure and guarantee a sufficient oxygen supply during slicing, which present advantages over the traditional use of a tissue chopper. Moreover, different cutting planes can be applied for vibratome brain slice preparations. Here, a detailed protocol for a successful preparation of vibratome-cut horizontal hippocampal slices of mouse brains is provided. In contrast to other slice preparations, horizontal slicing allows to keep the fibers of the hippocampal input path (perforant path) in a fully intact state within a slice, which facilitates the investigation of entorhinal-hippocampal interactions. Here, we provide a thorough protocol for the dissection, extraction, and acute horizontal slicing of the murine brain, and discuss challenges and potential pitfalls of this technique. Finally, we will show some examples for the use of brain slices in further applications.

This article aims to describe a systematic protocol to obtain horizontal hippocampal brain slices in mice. The objective of this methodology is to preserve the integrity of hippocampal fiber pathways, such as the perforant path and the mossy fiber tract to assess dentate gyrus related neurological processes.

شرائح فرس النهر الأفقية من دماغ الفأر

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as ...

Recording Whole-Cell Currents with GABA Puffing in a Mouse Brain Slice

Source: Feng, Y. et al., <a href="https://app.jove.com/v/56387">Whole-cell Currents Induced by Puff Application of GABA in Brain Slices.</a>&#160;J. Vis. Exp. (2017)
This video demonstrates the recording of whole-cell currents induced by gamma-aminobutyric acid, or GABA, in a mouse prefrontal cortical brain slice. Controlled delivery of GABA opens chloride channels in a postsynaptic neuron, generating inhibitory postsynaptic currents, or IPSCs. These IPSCs increase with higher GABA concentrations, reflecting GABA&#39;s inhibitory activity on synaptic transmission.

تسجيل تيارات الخلية الكاملة مع نفخ GABA في شريحة دماغ الفأر

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Preparation of Solutions

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JoVE Encyclopedia of Experiments

Neurophysiology

Electrophysiology Techniques

Visualization and Mapping of Neuronal Pathways in an Amygdala Brain Slice

Source: Bosch, D., et al. <a href="https://app.jove.com/v/53628">Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices.</a> J. Vis. Exp. (2016).
This video demonstrates a procedure for analyzing synaptic connectivity between medial prefrontal cortex (mPFC) and amygdala neurons using acute amygdala brain slices from mice. The mPFC neurons in these mice express channelrhodopsin fused to a fluorescent protein. The channelrhodopsins facilitate ion influx and action potential generation in the mPFC neurons upon light activation. Consequently, mPFC neurons release neurotransmitters that bind to postsynaptic amygdala neurons, triggering postsynaptic currents that are recorded to visualize mPFC-amygdala connectivity.

GABA تنشيط التيارات قناة واحدة وتونيك في شرائح الدماغ الجرذ

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excitotoxic المواد تحفيز الدماغ باعتبارها Microslices