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

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JoVE Journal

Neuroscience

25.0K 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

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.

Pair recordings are simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling precise electrophysiological and pharmacological characterization of the synapses between individual neurons. Here we describe the detailed methodology and requirements for establishing this technique in organotypic hippocampal slice cultures in any laboratory equipped for electrophysiology.

تقرن تسجيلات خلية كاملة في عضوي شرائح الحصين

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

Whole-cell Patch-clamp Recordings in Brain Slices

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the neuron. In this configuration, the micropipette is in tight contact with the cell membrane, which prevents current leakage and thereby provides more accurate ionic current measurements than the previously used intracellular sharp electrode recording method. Classically, whole-cell recording can be performed on neurons in various types of preparations, including cell culture models, dissociated neurons, neurons in brain slices, and in intact anesthetized or awake animals. In summary, this technique has immensely contributed to the understanding of passive and active biophysical properties of excitable cells. A major advantage of this technique is that it provides information on how specific manipulations (e.g., pharmacological, experimenter-induced plasticity) may alter specific neuronal functions or channels in real-time. Additionally, significant opening of the plasma membrane allows the internal pipette solution to freely diffuse into the cytoplasm, providing means for introducing drugs, e.g., agonists or antagonists of specific intracellular proteins, and manipulating these targets without altering their functions in neighboring cells. This article will focus on whole-cell recording performed on neurons in brain slices,&#160;a preparation that has the advantage of recording neurons in relatively well preserved brain circuits, i.e., in a physiologically relevant context. In particular,&#160;when combined with appropriate pharmacology, this technique is a powerful tool allowing identification of specific neuroadaptations that occurred following any type of experiences, such as learning, exposure to drugs of abuse, and stress. In summary, whole-cell patch-clamp recordings in brain slices provide means to measure in ex vivo preparation long-lasting changes in neuronal functions that have developed in intact awake animals.

This protocol describes basic procedural steps for performing whole-cell patch-clamp recordings. This technique allows the study of&#160;the electrical behavior of neurons, and when performed in brain slices, allows the assessment of various neuronal functions from neurons that are still integrated in relatively well preserved brain circuits.

تسجيلات خلية كاملة التصحيح، المشبك في الدماغ شرائح

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

تسجيل اللدونة متشابك في شرائح هيبوكامبال الحاد تحتفظ في صغيرة الحجم-إعادة التدوير، ونضح-، وغمر من نوع نظام الدائرة

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

Evaluation of Synapse Density in Hippocampal Rodent Brain Slices

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is tightly regulated during development and neuronal maturation. Importantly, deficits in synapse number can lead to cognitive dysfunction. Therefore, the evaluation of synapse number is an integral part of neurobiology. However, as synapses are small and highly compact in the intact brain, the assessment of absolute number is challenging. This protocol describes a method to easily identify and evaluate synapses in hippocampal rodent slices using immunofluorescence microscopy. It includes a three-step procedure to evaluate synapses in high-quality confocal microscopy images by analyzing the co-localization of pre- and postsynaptic proteins in hippocampal slices. It also explains how the analysis is performed and gives representative examples from both excitatory and inhibitory synapses. This protocol provides a solid foundation for the analysis of synapses and can be applied to any research investigating the structure and function of the brain.

A protocol for accurately identifying and analyzing synapses in hippocampal slices using immunofluorescence is outlined in this article.

تقييم كثافة المشبك في الدماغ القوارض هيبوكامبال شرائح

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is ...

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 ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain slices.

تصوير الكالسيوم اثنين-فوتون في Dendrites العصبية في الدماغ شرائح

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, very few have been widely accepted. Here, we propose&#160;a new procedure&#160;adapted from the well-known ex vivo brain slice technique, which offers a closer in vivo-like scenario than in vitro preparations, for investigating the early events triggering cell degeneration, as observed in Alzheimer&#39;s disease (AD). This variation consists of simple and easily reproducible steps, which enable preservation of the anatomical cytoarchitecture of the selected brain region and its local functionality in a physiological milieu. Different anatomical areas can be obtained from the same brain, providing the opportunity to perform multiple experiments with the treatments in question in a site-, dose-, and time-dependent manner. Potential limitations&#160;which could affect the outcomes related to this methodology are related to the conservation of the tissue, i.e., the maintenance of its anatomical integrity during the slicing and incubation steps and the section thickness, which can influence the biochemical and immunohistochemical analysis. This approach can be employed for different purposes, such as exploring molecular mechanisms involved in physiological or pathological conditions, drug screening, or dose-response assays. Finally, this protocol could also reduce the number of animals employed in behavioral studies. The application reported here has been recently described and tested for the first time on ex vivo rat brain slices containing the basal forebrain (BF), which is one of the cerebral regions primarily affected in AD. Specifically, it has been demonstrated that the administration of a toxic peptide derived from the C-terminus of acetylcholinesterase (AChE) could prompt an AD-like profile, triggering, along the antero-posterior axis of the BF, a differential expression of proteins altered in AD, such as the alpha7 nicotinic receptor (&#945;7-nAChR), phosphorylated Tau (p-Tau), and amyloid beta (A&#946;).

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

We present a method which can&#160;provide further insights into the early events underlying neurodegeneration and based on the established ex-vivo brain technique, combining the advantages of in vivo and in vitro experiments. Moreover, it represents a unique opportunity for direct comparison of treated and untreated group in the same anatomical plane.

وثمة نهج بديل لدراسة الأحداث الرئيسية في نيوروديجينيريشن باستخدام شرائح المخ الفئران السابقين فيفو

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, ...

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) ...

Assessment of Long-term Depression Induction in Adult Cerebellar Slices

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from parallel fibers (PF) to Purkinje cells (PC) is considered the basis for motor learning, and deficiencies of both LTD and motor learning are observed in various gene-manipulated animals. Common motor learning sets, such as adaptation of the optokinetic reflex (OKR), the vestibular-ocular reflex (VOR), and rotarod test were used for evaluation of motor learning ability. However, results obtained from the GluA2-carboxy terminus modified knock-in mice demonstrated normal adaptation of the VOR and the OKR, despite lacking PF-LTD. In that report, induction of LTD was only attempted using one type of stimulation protocol at room temperature. Thus, conditions to induce cerebellar LTD were explored in the same knock-in mutants using various protocols at near physiological temperature. Finally, we found stimulation protocols, by which LTD could be induced in these gene-manipulated mice. In this study, a set of protocols are proposed to evaluate LTD-induction, which will more accurately allow examination of the causal relationship between LTD and motor learning. In conclusion, experimental conditions are crucial when evaluating LTD in gene-manipulated mice.

In some gene-manipulated animals, using a single protocol may fail to induce LTD in cerebellar Purkinje cells, and there may be a discrepancy between LTD and motor learning. Multiple protocols are necessary to assess LTD-induction in gene-manipulated animals. Standard protocols are shown.

تقييم الحث علي الاكتئاب علي المدى الطويل في شرائح البالغين المخيخار

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from ...

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of pyramidal neurons and dendritic spines, a ballistic labeling technique can be utilized. In the present protocol, pyramidal neurons are labeled with DilC18(3) dye and analyzed using neuronal reconstruction software to assess neuronal morphology and dendritic spines. To investigate neuronal structure, dendritic branching analysis and Sholl analysis are performed, allowing researchers to draw inferences about dendritic branching complexity and neuronal arbor complexity, respectively. The evaluation of dendritic spines is conducted using an automatic assisted classification algorithm integral to the reconstruction software, which classifies spines into four categories (i.e., thin, mushroom, stubby, filopodia). Furthermore, an additional three parameters (i.e., length, head diameter, and volume) are also chosen to assess alterations in dendritic spine morphology. To validate the potential of wide application of the ballistic labeling technique, pyramidal neurons from in vitro cell culture were successfully labeled. Overall, the ballistic labeling method is unique and useful for visualizing neurons in different brain regions in rats, which in combination with sophisticated reconstruction software, allows researchers to elucidate the possible mechanisms underlying neurocognitive dysfunction.

We present a protocol to label and analyze pyramidal neurons, which is critical for evaluating potential morphological alterations in neurons and dendritic spines that may underlie neurochemical and behavioral abnormalities.

وضع العلامات البالستية للخلايا العصبية الهرمية في شرائح الدماغ وفي ثقافة الخلايا الأولية

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...